
WBHsm 

flBralilil 
lil81 |g|| 

Ml 

lli l fl l l 
Hnl 




ill 

ml 



m 



BBH 



! ; 9HHF 




nam HT&37 



Book 



AA \ L 



TO 



COPYRIGHT DEPOSm 



HOSPITAL LABORATORY METHODS 

FOR 

STUDENTS, TECHNICIANS AND CLINICIANS 



MCJUNKIN 



Hospital Laboratory Methods 

FOR 

Students 
Technicians and Clinicians 



BY 



FRANK A. McJUNKIN, A. M., M. D. 

PROFESSOR OF PATHOLOGY, MARQUETTE UNIVERSITY SCHOOL OF MEDICINE 
MILWAUKEE, WIS. 



WITH ONE COLORED PLATE AND 
NINETY-THREE ILLUSTRATIONS IN TEXT 



PHILADELPHIA 

P. BLAKISTON'S SON & CO, 

1012 WALNUT STREET 



-fi-8; 



Copyright, 1916, by P. Blakiston's Son & Co. 



r* 



MAY 31 1916 



THE MAPLE PRESS Y O K K FA. 



>CI.A431285 



PREFACE 

The purpose of this book is to place in the hands of the hospital 
laboratory worker and the technician of other small laboratories, 
directions for the tests that are commonly required on specimens 
sent to such laboratories. 

These specimens may be grouped as clinical laboratory (urine, 
gastric contents, feces, sputum, and blood), bacteriological, surgical 
and autopsy specimens. The manner in which the material should 
be handled before and after it reaches the laboratory is indicated in 
a specific way. There are usually several methods for obtaining 
the same results but in no case has an attempt been made to enumer- 
ate these; the simplest procedure by which the object of the analysis 
can be obtained with the greatest certainty has been chosen. No 
methods that are new and not thoroughly tried out have been 
given. 

The interpretation of. facts ascertained by chemical and cyto- 
logical examinations is very often possible only after consideration 
of the clinical condition of the patient. To place interpretations 
on laboratory findings, therefore, necessitates a thorough considera- 
tion of the various diseases and this has not been attempted. 

In our hands, the methods described below are used by the 
medical students taking clinical pathology and those electing a 
course in pathologic technic and by non-medical technicians to 
whom a course covering twenty-four weeks is offered during three 
consecutive summers. 

For whatever merit there is in these methods, 1 wish to acknowl- 
edge my indebtedness to Dr. F. B. Mallory of the Harvard Medical 
School under whom I received instruction while student and 
assistant in the laboratory of the Boston City Hospital. 



CONTENTS 



Page 

Urine i 

Introduction I 

Albumin I 

Heller's Test 2 

Heat and Acetic Acid Test 3 

Rough Estimation by Esbach Method 3 

Sugar 4 

Fehling's Test . 4 

Nylander's Test 4 

Phenylhydrazin Test 6 

Polariscopic Determination ■. 6 

Quantitative Determination by Fehling's Method 6 

Quantitative Determination by Fermentation . 7 

Acetone 7 

Diacetic Acid 8 

Oxybutyric Acid 8 

Bile Pigment 8 

Diazo-reaction 8 

Indican 9 

Chlorides 9 

Phthalein Test 9 

Total Nitrogen 11 

Urea Nitrogen 11 

Rough Determination of Urea by Ureometer 12 

Ammonia Nitrogen 13 

Uric Acid 13 

Sediment ■ . 13 

Unorganized Sediment of Acid Urine 14 

Unorganized Sediment of Alkaline Urine 16 

Organized Sediment 16 

Epithelial Cells -. 17 

Pus 17 

Blood 17 

Casts 17 

Cylindroids 17 

Mucous Threads 17 

Gastric Juice 18 

Introduction 18 

Fasting Stomach 18 

Bread- water Breakfast. 18 

vii 



Vlll CONTENTS 

Page 

Microscopic Examination 18 

Chemical Examination 19 

Gunzberg's Test 19 

Quantitative Titration 20 

Lactic Acid 21 

Pepsin 22 

Rennin 22 

Starch 22 

Meat Dinner 22 

Feces 22 

Introduction 22 

Undigested Food Particles 23 

Substances Escaping from Biliary Tract 23 

Occult Blood 24 

Parasites 24 

Bacteria 24 

Tubercle Bacilli 24 

Dysentery Bacilli 24 

Bacilli of Typhoid Colon Group 24 

Ameba • 25 

Entameba histolytica 25 

Entameba coli 25 

Flagellates 25 

Cercomonas hominis 25 

Trichomonas intestinalis 25 

Lamblia intestinalis ..." 25 

Infusoria 26 

Balantidium coli 26 

Nematodes 26 

Uncinaria americana 26 

Ankylostoma duodenale 26 

Strongyloides stercoralis 27 

Tricocephalus dispar 28 

Ascaris lumbricoides 28 

Trichinella spiralis 28 

Trematodes 28 

Schistosoma hematobium 28 

Cestodes 28 

Tenia saginata 28 

Tenia solium 28 

Dibrothrocephalus latus 28 

Tenia nana 28 

Sputum 29 

Introduction 29 

Gross Examination 29 

Fresh Microscopic Examination 30 



CONTENTS IX 

Page 

Stained Preparations 30 

Ziehl-Neelsen Stain 30 

Gram's Stain 32 

Blood Stain 32 

Blood 32 

Introduction 32 

Hemoglobin 32 

Tallquist Scale Method 33 

Sahli Hemometer Method 34 

Miescher Hemoglobinometer method 35 

Dare Hemoglobinometer method 37 

Enumeration of the Red Blood Corpuscles 37 

Enumeration of the White Blood Cells 40 

Coagulation Time of the Blood 40 

Blood Films 41 

Differential Counting of the Leucocytes 42 

Lymphoblastic Cells. 43 

My eloblastic Cells 46 

Endothelial Leucocytes 47 

Red Blood Corpuscles ' 48 

Platelets 48 

Protozoa in Blood Films 48 

Mcjunkin's Polychrome Stain for Protozoa 48 

Widal Reaction 50 

Phagocytosis in Vitro 50 

Blood Cultures 52 

WassermannTest 55 

Introduction 55 

Sera 56 

Complement 58 

Amboceptor 58 

Antigen 59 

Cholesterin Antigen 60 

Acetone-insoluble Antigen 60 

Gonococcus Antigen 61 

Testing the Antigens . 61 

Hemolytic Property 61 

Anticomplimentary Property 62 

Antigenic Property 62 

Corpuscles 63 

Saline 64 

Test 64 

Titration for Amboceptor Unit 64 

Technic for Actual Test 65 

Antigen-complement-amboceptor Table 66 

Bacteriological Specimens 68 



X CONTENTS 

Page 

Introduction (38 

Media 72 

Plating 72 

Tube Cultures 72 

Agar 73 

Bouillon 75 

Litmus Milk 76 

Gelatin 76 

Dunham's Solution 77 

Serum Water . m 77 

Loeffler's Blood Serum 77 

Drigalski Medium 77 

PetrofT Medium for Isolation of Tubercle Bacilli 78 

Stains . '. 79 

Loeffler's Methylene Blue. . 79 

Gram's Stain 79 

Ziehl-Neelsen's Stain for Tubercle Bacilli 80 

Capsule Stain ■ 82 

Flagellum Stain 82 

Spore Stain . . 82 

Special Specimens. . 82 

Cerebro-spinal Fluid 83 

Fluid for Tubercle Bacilli 83 

Pus or Curettings for B. Aerogenes Capsulatus 84 

Material for Tetanus 84 

Milk 85 

Nose and Throat Cultures for Diphtheria Bacilli 85 

Bacterial Vaccines 87 

Surgical Specimens 88 

Introduction 88 

Frozen Sections for Immediate Diagnosis 89 

Celloidin Sections 91 

Hematoxylin-eosin Stain for Celloidin Sections 97 

Paraffin Sections 98 

Zenker-nuid Fixation 99 

Decalcification of Bone 104 

Formalin Fixation 104 

Alcohol Fixation * 106 

Cutting Paraffin Sections 107 

Eosin-Methylene Blue Stain (Mallory) 109 

Phosphotungstic Acid Hematoxylin Stain (Mallory) 112 

Aniline Blue Stain (Mallory) 112 

Elastic Fibril Stain (Verhoeff) 112 

Scharlach R Stain for Fat 113 

Hemosiderin Reaction ' 113 

Calcium Reaction 114 



CONTENTS XI 

Page 

Glycogen Stain 114 

Gram-Weigert Stain 114 

Treponema Pallidum 114 

Mcjunkin's Stain for Bacteria and Protozoa 115 

Tubercle Bacillus Stain 115 

Leprosy Bacillus Stain 115 

Myelin Sheath Stain 117 

Degenerated Sheath Stain 117 

Ganglion Cell Stain 117 

Chromaffin Cell Stain 118 

Autopsy Specimens 118 

Introduction 118 

External Body Surfaces 119 

Peritoneal Cavity 120 

Pleural Cavities 121 

Pericardial Cavity 122 

Heart 122 

Lungs 122 

Spleen 123 

Gastro-intestinal Tract 124 

Pancreas 124 

Liver . 124 

Kidneys 124 

Adrenals 125 

Genito-urinary Organs 125 

Aorta 125 

Neck Organs 125 

Brain 126 

Middle Ear 127 

Naso-pharynx 127 

Bone Marrow 127 

Spinal Cord . 127 

Fixation of Autopsy Specimens 127 

Museum Preparations 130 

Index 133 



HOSPITAL LABORATORY METHODS 



URINE 



Introduction. — Each hospital patient has a 3-liter, narrow- 
mouthed bottle provided with a funnel and containing 10 c.c. of 
toluol. Chloroform has been widely used for preserving urine for 
chemical examination but it cannot be entirely driven off. by heat. 
As soon as the urine is voided it is emptied into this bottle and 
the total amount passed during the twenty-four-hour period 
is collected. This twenty-four-hour sample is used for chemical 
analysis. The sample for microscopic examination is sent to 
the laboratory in a pint clamp-stoppered tonic or beer bottle 
within an hour after it is voided. If the sample for microscopic 
purposes cannot be sent to the laboratory soon after it is voided a 
small amount of 10 per cent, formalin may be added. Urine to 
which formalin has been added is not suitable for chemical analysis. 

Normal urine when voided is usually clear; rarely in strongly 
alkaline urine there is a cloudiness due to precipitated phosphates 
and carbonates. Soon a cloud (nubecula) is formed by the collec- 
tion of epithelial cells on the mucin present. The color varies from 
colorless to dark amber. The amount is from 800 c.c. to 3000 c.c. 
An alkaline reaction in the fresh urine is due to alkaline (disodium) 
phosphates and to carbonates, while the acid reaction usually present 
results from an excess of dihydrogen phosphates. There may be 
such a combination of these phosphates that the reaction is am- 
photeric. The specific gravity is taken with a urinometer (Fig. 1). 
It has a normal variation between 1.010 and 1.025. 

Albumin. — The usual abnormal protein substances present are 
serum albumin and serum globulin but nucleoalbumin, mucin and 
albumose may be present. Resins, uric acid, urates, urea and the 
bile acids may with some tests react like albumin. If the urine is 



HOSPITAL LABORATORY METHODS 



not clear, filter it before testing for albumin or add kieselguhr and 
filter. 

Heller's Test (Fig. 2). — Place 5 c.c. concentrated nitric acid 
(a) in a test-tube and stratify the urine (c) above the acid by slant- 
ing the test-tube and allowing the clear urine to 'trickle down its side 





Urinometer. 



Fig. 2. 
Fig. 2. — Urine. Heller's Test, a, Nitric acid; b, precipitate at surface of con- 
tact; c, filtered urine; d, filter paper; e, 5-cm. funnel. The test-tube is heavy glass 
with a non-flaring mouth 155 by 16 mm. outside measurements. A medium-size 
tube for Wassermann work, etc., measures 120 by 13 mm. A small serum tube 
measures 55 by 7 mm. These test-tubes are obtained from Bausch & Lomb 
Optical Co. 

from the funnel (e) while filtering. A white ring (6) at the surface 
of contact may be due to albumin. When the Heller test is posi- 
tive, protein substances other than albumin must be ruled out by 
further observations and tests. Nucleoalbumin and mucin produce 



URINE 3 

a ring several millimeters above the surface of contact and on gentle 
agitation this clouding ascends. If due to albumose, on applying 
gentle heat the ring disappears. Therefore, to rule out nucleo- 
albumin, mucin and albumose in all cases in which the ring is 
obtained, agitate and then apply gentle heat. 

As regards non-protein substances, if resins are suspected add a 
few drops of concentrated hydrochloric acid to 10 c.c. of the original 
urine and shake. Resins are precipitated. The remaining sub- 
stances, uric acid, urates, urea and the bile acids, do not give the test 
unless the urine is concentrated. Of these substances urates most 
commonly give a precipitate. A precipitate due to urates disap- 
pears on heating. 

Heat and Acetic Acid Test. — If a ring is obtained with Heller's 
test, the heat and acetic acid test should be applied. To 20 c.c. 
urine in a test-tube, add 5 drops 20 per cent, acetic acid and 5 c.c. of 
a saturated solution of sodium chloride. Boil the upper half of the 
contents of the tube, using the lower half as a control. The sodium 
chloride keeps the nucleoalbumin and the mucin in solution, while 
albumose is soluble in the heated solution. Uric acid, urates, urea 
and the bile acids do not precipitate. If a precipitate of resins is 
suspected, cool and shake with 10 c.c. of benzol. Any precipitate 
of resins goes into solution in the benzol. 

Albumose (and Bence- Jones body) form a coagulum at about 
6o°C. in the heat and acetic acid test but the precipitate almost 
entirely disappears when the boiling point is reached. 

Rough Estimation of Albumin by Esbach Method.— Fill the 
albuminometer (Esbach obtained from Spencer Lens Co., Buffalo, 
N. Y.) to the mark "U" with acidified urine and to the mark "R" 
with Esbach's reagent (picric acid 1, citric acid 2, and distilled water 
100). Stopper, thoroughly mix by inverting and set aside in a 
test-tube rack for twenty-four hours. The reading on the tube that 
corresponds to the upper surface of the precipitate is the number of 
grams per liter of protein. Less than 0.5 gm. per liter will not 
sediment. This determination of protein is not accurate and has a 
doubtful value. 

If an accurate quantitative estimation of albumin is desired, the 



4 HOSPITAL LABORATORY METHODS 

nitrogen of the proteins may be determined by taking the difference 
between the total nitrogen and the nitrogen after the precipitation 
and removal of all proteins present. 

Sugar (Glucose). — About }i per cent, glucose is normally 
present in the urine. This amount does not react with the usual 
tests. Levulose gives the same reduction tests as glucose, and when 
present the polariscopic and reduction quantitative estimates do not 
check. Maltose has 2.5 times as great dextrorotatory power as 
glucose and may therefore produce a discrepancy in the polariscopic 
and other quantitative estimations. Levulose and maltose when 
present are usually accompanied by glucose and have the same sig- 
nificance. Lactose may be present in the urine of lactating women. 
Lactose is not fermented by yeasts and reduces copper only slowly. 
Pentoses are rarely present. They reduce copper slowly and are 
inactive optically. Glycuronic acid conjugated with other sub- 
stances such as indoxyl occurs in normal and pathologic urines. 
With Fehling's test they give a greenish reduction. If there is a 
suspicion of glucose, make the phenylhydrazin test. With this 
test no crystals are obtained with the glycuronates. Homogentisic 
acid which is present in the urine of alkaptonurics reduces Fehling's 
solution, but is readily recognized owing to the urine turning brown 
and then black on standing exposed to the air. 

Fehling's Test. — If the urine is not clear, add lead acetate 
and filter. Remove albumin by the heat and acetic acid method. 
Place 5 c.c. Fehling's solution A (copper sulphate, Merck reagent, 
34.65 gm., distilled water 1000 c.c.) and 5 c.c. Fehling's solution B 
(Rochelle salt 173 gm., sodium hydrate 125 gm., and distilled water 
1000 c.c.) in a test-tube and boil. Remove from flame and at once 
add about 1 c.c. urine and allow a few minutes for reduction to 
take place. A reduction is shown by the appearance of a brick red 
precipitate. 

Nylander's Test. — To prepare Nylander's reagent dissolve 
4 gm. Rochelle salt in 100 c.c. of warm 10 per cent, sodium hy- 
drate, add 2 gm. bismuth subnitrate, shake, and filter into a 
dark-colored bottle. To 10 c.c. urine from which all albumin has 
been removed, add 1 c.c. of the reagent and place in boiling water 



URINE 



in a water-bath for live minutes. If positive, the urine darkens 
and the black precipitate settles out during the heating. Uric 
acid, creatinin, and the alkaptone bodies may produce a dirty 





Fig. 3. — Urine. Quantitative Fehling's Test, a, White base or base covered 
with filter paper; b, burette containing the diluted urine; c, asbestos gauze; d, 
solution; examine for the clear zone, e, against a white background after removing 
from the flame;/, folded muslin to be held with thumb and finger. 



yellow color (atypical reaction) with Fehling's test but do not 
react to Nylander's test. Chloroform must be removed before 
applying tests for sugar. Urine to which formalin has been added 
must never be used. 



6 HOSPITAL LABORATORY METHODS 

Phenylhydrazin Test. — In cases in which the reduction is not 
altogether typical this test should be applied. To 5 c.c. urine in a 
test-tube add 5 drops of a pure phenylhydrazin base (Merck & 
Co.) and 10 drops of glacial acetic acid. Boil one minute. Add 
5 drops 15 per cent, sodium hydroxide and heat again for a few 
seconds and then set aside to cool. The yellow sheaf-like crystals 
appear at once or within twenty minutes. The crystals obtained 
from the bottom of the tube with a pipette are examined under 
the high dry objective. If the specific gravity of the urine is 
high the sample should be diluted until it is below 1.020. 

Polariscopic Determination of Glucose.— Two grams of lead 
acetate are added to the twenty-four-hour sample of urine. After 
filtering, fill the 189.4 mm. glass tube with the clear urine, apply 
the glass disk, being careful not to get an air bubble beneath it, 
and screw on the metal cap. Place the tube in the polariscope and 
starting at zero turn the handle until the field is equally illumin- 
ated. The percentage of sugar is read off from the vernier 
provided the instrument is made for sugar estimation only 
(saccharometer) . 

Quantitative Determination of Glucose by Fehling's Method. — 
Place 10 c.c. (accurate) Fehling's A (10 c.c. of Fehling's^ contains 
0.3465 gm. copper sulphate) and 10 c.c. of Fehling's B (approxi- 
mate) in a 300-c.c. Florence flask of Jena glass (Fig. 3) and dilute 
with about 50 c.c. of water. Heat to boiling and run in less than 
1 c.c. of the diluted (If the specific gravity of the urine is 1.030 or 
over dilute it 1-10 with water; if under 1.030 dilute 1-5 with water.) 
urine from a burette (b) and again boil. Repeat this until no 
blue color remains. Examine for this end point (absence of 
bluish tinge) by looking against a white sheet of paper on the 
clear line (e) 3 mm. below the top of the meniscus. A yellowish 
change in the red precipitate indicates that too much urine has 
been added: 0.05 gm. of glucose are required for the reduction of 
this amount of Fehling's. For example if 8 c.c. of a 1-5 dilution 
is required to discharge the blue, 8 c.c. of this dilution or 1.6 c.c. 
of the undiluted urine contains 0.05 gm. glucose; 1 c.c. of the 
undiluted urine 0.032 gm.; 100 c.c. contains 3.2 gm. or 3.2 per cent. 



URINE 7 

A simple method for computing dilutions should be used. 
By a dilution one in ten (i-io) is meant such a dilution that in 
10 parts of it there is i part of the original liquid. To make 
such a dilution take i part of the original liquid and 9 parts 
of water. More complicated dilutions are often required; for 
example, from a 14 per cent, solution (14-100), make a 3 per cent, 
solution (3-100). One part of the 14 per cent, solution contains 
1 ^1loo P art of the original liquid/ while 1 part of the required 3 
per cent, solution contains %oo P ar t of the original solution. 
Therefore the 14 per cent, solution is ^{oo divided by ^00 ° r 
4.66 times too strong. Therefore take 1 part of the 14 per cent, 
solution and the remainder of water which is 3.66 parts. 

Quantitative Determination of Glucose by Fermentation. — 
Dilute the urine so that the specific gravity is below 1.008 and the 
percentage of sugar below 1. Fill with the diluted urine the 
test-tube that comes with the Einhorn saccharometer (obtained 
from Spencer Lens Co., Buffalo, N. Y.) to the mark and add a 
knife-point of fresh compressed yeast. Shake well, fill the sac- 
charometer displacing all air in the closed end and place in the 
incubator for twenty-four hours. The percentage of sugar is 
indicated on the blind arm by the amount of gas present. Multi- 
ply this by the dilution made to get the percentage of sugar. 

This method gives only an approximate result and should not 
be used in careful work for quantitative determinations. 

Acetone (Lange).- — To 20 c.c. urine add 1 c.c. of a 10 per cent, 
solution of sodium nitroprusside in 10 per cent, acetic acid. Mix, 
and then stratify a few cubic centimeters of strong ammonia on the 
surface. If a violet color appears at the surface of contact and 
persists, it is most likely due to acetone. To stratify any liquid 
slant the tube (Fig. 2) and carefully let the lighter liquid flow 
down the side of the inclined tube. 

Acetone (Gunning). — Place 100 c.c. of urine in an extra large 
test-tube or in a small Erlenmeyer flask and add 1 c.c. concentrated 
hydrochloric acid. Stopper with a rubber cork carrying a glass 
tube 2 feet long curved at an angle of 45 degrees. Boil and collect 
the first 10 c.c. of distillate in a test-tube. To 5 c.c. of this add 



8 HOSPITAL LABORATORY METHODS 

5 drops of ammonia and iodine solution (iodine 4 gm., potassium 
iodide 6 gm. and water 100 c.c.) until the black precipitate formed 
does not immediately dissolve. If acetone is present a yellow pre- 
cipitate of iodoform soon forms. Examine this precipitate for 
hexagonal crystals using the high dry lens. A positive test is pro- 
duced only by acetone. 

Diacetic Acid. — To 10 c.c. urine, add 10 drops 10 per cent, 
ferric chloride solution and filter. The excess of phosphates is 
removed by the filtration. Now add a few drops more of the 
ferric chloride solution. If diacetic is present a red color appears. 
Place 10 c.c. urine in a second test-tube, boil for several minutes 
and proceed with the test exactly as above. If the red color was 
due to diacetic acid, it should be much less intense or entirely 
absent in the boiled urine. 

Oxybutyric Acid (Black).- — Evaporate 10 c.c. urine in a small 
porcelain dish to about one-third, acidify with a few drops of 
hydrochloric acid and add with a spatula plaster of Paris to a thick 
paste. When it begins to set break up into a meal with the 
spatula, add 30 c.c. ether and mix. Decant the clear ether (20 c.c.) 
into a dry evaporating dish, evaporate the ether over a water-bath 
and dissolve the residue in water (10 c.c). Neutralize with an 
excess of dry barium carbonate (about 12 gm.), pour into a 
test-tube, add a few drops of hydrogen peroxide and 5 drops of 
10 per cent, ferric chloride. A red color indicates oxybutyric 
acid. 

Bile Pigment (Gmelin). — Stratify the urine above concentrated 
nitric acid. If bile pigment is present a greenish color appears at 
the point of contact. 

To confirm this test for bile pigment, dilute tincture of iodine 
with 95 per cent, alcohol until it is straw-colored and then stratify 
the iodine solution on the urine. A greenish ring at the contact 
indicates bile pigment (Smith). 

Diazo-reaction (Ehrlich). — To 10 c.c. sulphaniiic acid solution 
(sulphalic acid 5, hydrochloric acid 50, and water 1000), add 0.5 
c.c of 0.5 per cent, sodium nitrite solution and 10 c.c. urine. 
Mix and then stratify with concentrated ammonia. If positive 



URINE 9 

'a pink ring forms at the surface of contact and when the mixture is 
shaken the foam appears pink. 

Indican (Jaffe). — To 2 c.c. chloroform in a test-tube add 5 c.c. 
urine, 5 c.c. concentrated hydrochloric acid, and 2 drops of a 
strong calcium hypochlorite suspension( 10 gm. in 100 c.c. water). 
Shake. A blue color in the chloroform is positive. Iodides may 
give a confusing red to purplish color. If they are suspected add a 
few drops of 10 per cent, sodium hyposulphite solution and shake. 
This removes any color in the chloroform due to iodides. 

Chlorides (Volhard). — To 5 c.c. urine (albumin-free) in a 
small Erlenmeyer flask add 20 c.c. distilled water, 10 c.c. silver 




Fig. 4. — Urine. Tubes for phthalein test, a, Narrow wooden rack containing 
ten test-tubes b, 155 by 16 mm. These tubes contain standard solutions of phenol- 
phthalein. Stopper with small corks. The top part of the rack c should be within 
one inch of the top of the tubes. 



nitrate solution (29.042 gm. silver nitrate in 1 liter of water), 
and 2 c.c. indicator (30 c.c. water + 70 c.c. nitric acid, specific 
gravity 1.2 + excess of ferric ammonium sulphate: filter). Now 
run in ammonium sulphocyanate (3.25 gm. ammonium sulpho- 
cyanate + 250 c.c. distilled water — 1 c.c. of this solution must be 
equivalent to 1 c.c. of the silver solution) until the first red appears 
and persists when stirred. 1 c.c. of the silver solution is equivalent 
to 0.01 gm. sodium chloride. 

Phthalein Test (Rowntree and Geraghty) (Fig. 4). — Have 
the patient drink a pint or more of water and catheterize the 



IO 



HOSPITAL LABORATORY METHODS 



bladder. Then inject intramuscularly i c.c. of a solution con- 
taining 6 mg. of phenolsulphophthalein. To 0.6 gm. phenol- 
sulphophthalein add 1 c.c. of N-sodium hydrate and 100 c.c. 
saline. Of this solution give 1 c.c. Hynson, Wescott & Co., 
Charles St., Baltimore, place the preparation on the market in 
1 c.c. ampules. The catheter is left in place and the urine 
allowed to flow into a container in which a drop of strong sodium 




*T*-*f*W 



a 



Fig. 5. — Urine. Folin apparatus for nitrogen determinations, a, Three pairs 
microburners under three pairs digestion tubes, c, that are provided with fume 
absorbers, d\ b, three pairs of tubes into which ammonia is aspirated; e, pipette 
for alkali; /, pinch cock in rubber connections; g, wire gauze covered with asbestos 
in which are holes for bottom of tubes; h, i and /, are boards. 



hydrate has been placed until the first red color appears (ten 
minutes normally) when the catheter is removed. At'the end of 
the first hour the patient urinates into a clean container, and at the 
end of a second hour into a second container. Place the first- and 
second-hour specimens in two separate 1000 c.c. cylinders and add 
distilled water to 1000 c.c. Make each alkaline. Compare these 



URINE 



II 




with standard samples in ten tubes (Fig. 4) containing 0.1, 0.2, 
0.3, 0.5, i.o, 2, 3, 4, 5, 6 mg. per 1000 c.c. 

Total Nitrogen in Urine (Folin and Farmer) (Fig. 5) . — Place 
5 c.c. urine in a 50-c.c. graduated flask and dilute up to volume. 
To 1 c.c. of diluted urine measured into a 25 X 200-mm. test-tube 
(Jena) (c) provided with a fume absorber 
(Fig. 6) add 1 c.c. sulphuric acid by means of 
a piette, 1 gm. potassium sulphate, 1 drop 10 
per cent, copper sulphate and a glass bead. 
Boil over a micro-burner for five to ten 
minutes to complete digestion with the fume 
absorber (d) in place. Cool until viscous, 
detach the fume absorber, and slowly add 6 
c.c. of water. Add 3 c.c. of 40 per cent. 
NaOH by drawing it into the pipette (e) 
containing 1 drop caprylic alcohol and hold- 
ing it there by means of a pinch cock (f) 
until the corks are inserted for aspiration 
into the front tubes (6). The suction is 
now started slowly at first and then con- 
tinued more rapidly for fifteen minutes in 
order to aspirate all the ammonia into the 
front tubes (b) which contains 10 c.c. one- 
fiftieth normal HO, 3 drops of a 1 per cent, 
alizarine red solution and 30 c.c. water. At 
the end of fifteen minutes wash the delivery 
tube and titrate the excess of acid in the 
tubes (b) with one-fiftieth normal NaOH (end 
reaction is a red color). One cubic centi- 
meter of one-fiftieth normal acid neutralized by the aspirated 
ammonia is equivalent to 0.00028 gm. nitrogen. Specimens are 
always examined in duplicate. It is convenient to have a rack 
with six tubes in the rear for the digestion and six in front for 
the ammonia absoprtion. With such an apparatus three separate 
determinations can be made at the same time. 

Urea Nitrogen. — Measure into tube (6), Fig. 5, 25 c.c. of one- 




Fig. 6. — Urine. Folin 
fume absorber. Made 
by Eimer and Amend or 
it may be blown from 
suitable glass tubing. 



12 HOSPITAL LABORATORY METHODS 

fiftieth normal hydrochloric acid, 3 drops of alizarin red and 1 
drop of caprylic alcohol. In tube (c) place 5 c.c. of urine diluted 
1-10, 1 c.c. of 15 per cent, urease in water (the urease is obtained 
from Arlington Chemical Co., Yonkers, N. Y.) and 1 drop of 
caprylic alcohol. Stopper (c) and (b) and allow to stand fifteen 
minutes then pass air for one-half minute to remove any ammonia 
fumes in the upper part of the tube. Add 5 gm. potassium 
carbonate to (c) and pass air for fifteen minutes. Each cubic 
centimeter of one-fiftieth normal acid neutralized is equivalent 
to 0.00028 gm. nitrogen. Find the number of grams of nitrogen 
that would be liberated from 100 c.c. of urine. by this test. From 
this amount subtract the number of grams of ammonia nitrogen 
in 100 c.c. of the urine and the remainder will be the number of 
grams of urea nitrogen in 100 c.c. of the urine. 

Rough Determination of Urea by Ureometer. — Add to the 
small side tube of the ureometer (Doremus-Hinds obtained from 
Spencer Lens Co., Buffalo, N. Y.) 1 c.c. urine allowing it to flow 
in through and fill the opening in the glass cock. Close the cock 
and wash out with water any urine that has run into the bulb 
portion of the instrument, after which it is ready for the addition 
of the hypobromite solution. 

To prepare the hypobromite solution place in a large test- 
tube 13 c.c. Rice's solution A (bromine 10 c.c, potassium bromide 
31 gm., distilled water 250 c.c.) and 15 c.c. of Rice's solution B 
(sodium hydrate 100 gm., and distilled water 250 c.c). Pour 
this into the bulb portion of the apparatus incline so as to displace 
the air from the blind arm, completely filling it with the hypo- 
bromite mixture. Fill the small side tube with urine and allow 
exactly 1 c.c. of urine to flow slowly into the bulb portion. Each 
large division of the blind arm represents 1 per cent. urea. The 
large divisions are divided into tenths. For example, if the column 
of gas collecting in the blind arm measures 0.014, then the per- 
centage of urea would be 1.4. 

The determination of urea by this method when most care- 
fully carried out is not an accurate one and it has a doubtful 
use. 



URINE 13 

Ammonia Nitrogen (Folin-Macallum).- — Place 2 c.c. urine in 
the rear tube and add 0.5 c.c. ammonia reagent (15 gm. potassium 
carbonate, 15 gm. potassium oxalate, water 100 c.c.) and 2 drops 
of caprylic alcohol. Connect up at once and proceed as in the 
total nitrogen determination. 

Uric Acid (Folin and Shaffer). — In each of two beakers place 
150 c.c. fresh urine and add to each 37.5 c.c. Folin-Shaffer reagent 
(500 gm. ammonium sulphate, 5 gm. uranium acetate, 60 c.c. of 
10 per cent, acetic acid and 500 c.c. of distilled water). Stir 
vigorously and filter after a few minutes. Measure 125 c.c. of the 
filtrate (100 c.c. of urine) into a beaker and add exactly 5 c.c. 
concentrated ammonia, cover the beaker and allow to stand 
forty-eight hours. At the end of this time filter off the ammonium 
urate and wash several times with 10 per cent, ammonium sul- 
phate. Now wash the precipitate back into the beaker with 
exactly 100 c.c. of distilled water and 20 c.c. of concentrated 
sulphuric acid and titrate immediately with one-twentieth normal 
potassium permanganate (1.5780 gm. per liter) until the first 
permanent pink color appears. Each cubic centimeter of per- 
manganate equals 3.76 mg. of uric acid. The two samples must 
check. 

Sediment. — To obtain sediment for microscopic examination, 
freshly voided urine is centrifuged. The sediment is taken from 
the bottom of the centrifuge tube with a small pipette, transferred 
to a slide and examined with the low power. If necessary drop a 
cover on the sediment and examine with the high dry. A reagent 
may be drawn under one side of the cover by applying filter paper 
to the opposite side. In examining unstained objects cut the 
light down to a minimum. The Abbe condenser may be swung 
out to advantage. 

Surgical Urines. — The urine should be examined before a 
general anesthetic is administered. In hospitals where there are 
very large surgical wards these examinations may become burden- 
some. In such cases rather than carelessly examine the urine the 
above technic should be altered somewhat. Urine cylinders with 
a cone at the bottom (obtained from Arthur Thomas and Co., 



14 • HOSPITAL LABORATORY METHODS 

Philadelphia) are placed on a table and all the urine emptied into 
these before beginning the tests. Remove the slip from the 
bottle and place it under the cylinder at the time the urine is poured 
into it. To test for albumin use a modified Heller technic by 
substituting a piece of glass tubing a foot long with a 3-mm. 
lumen for the test-tube. Place the tubing in the urine in the 
cylinder to a depth of 1 inch and then retain the urine by firmly 
pressing the thumb over the top. Lower into a graduate con- 
taining concentrated nitric acid until 1 inch of the acid is forced 
into the tube. Now remove the pipette from the acid with the 
thumb firmly on the top and examine for a white ring at the con- 
tact. On lowering the tube into the nitric do not remove the 
thumb from the top until it is lowered to the level of the urine in 
the pipette. 

To test for sugar, add a few drops from each of about five urines 
and perform the Fehling's test as usual. If positive then go back 
and determine which urine gave the positive test. 

To obtain sediment from the bottom of the cone, lower the 
glass tubing to the bottom holding the thumb over the top and 
then allow the desired amount of sediment to flow into it. Place 
a drop of the sediment on a glass slide large enough for at least 
five sediments. This method of examination should be used 
only when a more careful examination is prohibitive. 

The Unorganized Sediment of Acid Urine (Fig. 7). — In acid 
urine, amorphous urates (quadriurates of sodium and potassium) 
separate out on cooling and absorb urochrome (yellow) and uroery- 
thrin (red) from the urine. They dissolve on heating. Hydro- 
chloric acid dissolves amorphous urates and later uric acid crystals 
form. Uric acid, usually as whetstones, may separate out from 
the urine without the addition of acid. The uric acid crystals 
are insoluble in hydrochloric acid and soluble in mineral acids 
and insoluble in acetic. Calcium oxalate usually forms octahedra 
that are soluble in mineral acids. Cholesterin forms rhombic 
plates that turn carmine and then violet on the addition of sul- 
phuric acid. Hematoidin in icteric urine may form needles or 
rhombs. They are colored but do not react like uric acid. Cystin 




i5 



Fig. 7. — Urine. Unorganized sediment of acid urine, a, Amorphous quadri- 
urates of potassium and sodium; b, uric acid; c y calcium oxalate; d, monocalcium 
phosphate; e, calcium sulphate;/, cholesterin; g, cystin; h, hematoidin. 




Fig. 8. — Urine. Unorganized sediment of alkaline urine, a, Tricalcium and 
trimagnesium amorphous phosphates; b, amorphous calcium carbonate; c, ammo- 
nium-magnesium phosphate; d, ammonium biurate; e, dicalcium or dimagnesium 
phosphate. 



i6 



HOSPITAL LABORATORY METHODS 



occurs as colorless hexagonal plates that readily dissolve in hydro- 
chloric acid and in ammonia. 

The Unorganized Sediment of Alkaline Urine (Fig. 8). — In 
alkaline urine tricalcium and trimagnesium phosphates (amor- 
phous phosphates) form white deposits soluble in acids. Calcium 
carbonate is like these except that the addition of acid to it causes 
the evolution of gas. 




,--/' 



/ k 

Fig. 9. — Urine. Organized sediment, a, Squamous epithelium from ureter, 
bladder, urethra, prepuce, vulva or vagina; b, endothelial leucocyte with blood 
pigment; c, red blood corpuscle; d, neutrophile (pus cell); e, round epithelial cell 
from the small ducts of the genital apparatus; g, coarse granular cast; h, fine granular 
cast; i, mucous thread; j, cylindroid; k, waxy cast; ;;/-, pus cast; n t epithelial cast; 
0, blood cast. Examine with high dry under cover-glass after preliminary examina- 
tion with low power without cover. 

Ammonium magnesium phosphate (triple phosphate) is the 
common "coffin lid" crystal of alkaline urine. Ammonium 
biurate forms yellowish spheres or "morning star" crystals. 
Rarely dicalcium and dimagnesium phosphates form prisms in 
the urine. 

Organized Sediment (Fig. 9). — Tissue cells and debris are 
spoken of as organized sediment in distinction to unorganized 
amorphous and crystalline sediment. 



URINE 1 7 

Epithelial Cells. — It is not at present possible to name the 
source of all epithelial cells from their morphologic appearance. 
Numerous small round cells when occurring with albumin may, 
perhaps, be considered as coming from the tubules of the kidney. 
It must be remembered that small round cells also come from small 
ducts of the male genitals. The ureter, bladder, urethra, prepuce, 
vulva, and vagina desquamate squamous epithelium. 

Pus. — Neutrophiles (pus cells) have finely granular cyto- 
plasm with polymorphous nuclei that are well brought out by 
allowing 3 per cent, acetic to flow under the cover. The presence 
of casts may indicate that the pus comes from the kidney. The 
source of the pus is best cleared up by cystoscopic examination 
and ureteral catheterization. Contamination of the specimen 
with leucorrheal discharge must be eliminated. Endothelial 
leucocytes containing blood pigment may be found in the urine. 

Blood. — Red blood corpuscles appear under the high dry lens 
as typical disks or only as shadows. Menstrual blood must be 
excluded. A test for chemical blood (guaiac) (see p. 24) is often 
desirable. 

Casts. — They are formed by the collection of foreign substances 
in the tubules of the kidney. Epithelial casts are made up largely 
of dead desquamated epithelium from the tubules. Pus casts 
are formed by the fusion of neutrophiles collected in the tubules. 
Blood casts are clots of blood formed in the tubules. Hyaline 
casts are formed by the discharge of hyaline droplets from the 
kidney cells. Hyaline casts are homogeneous. Waxy casts are 
more refractive than the hyaline casts, show fissures and fre- 
quently give the amyloid reaction with iodine. Epithelial, pus, 
and blood casts may form coarsely and finely granular casts 
when degeneration occurs and the cells disintegrate. Cylin- 
droids are hyaline in appearance but one end tapers off into a 
thread. They are said to have the same significance as hyaline 
casts. 

Mucous threads form the nubecula and are often present as 
twisted bands. In distinction to casts they are insoluble in acetic 
acid. 



l8 HOSPITAL LABORATORY METHODS 

"Clap" threads are made up of heavy mucous material that 
has collected many pus or epithelial cells. They are macroscopic. 

GASTRIC JUICE 

Introduction. — Vomitus may serve for the demonstration of 
blood but its examination is otherwise of little value. Suitable 
material for chemical and microscopic examination is obtained 
by passing the stomach tube. This is done in the morning before 
any food or drink has been taken (fasting stomach), one hour after 
a test breakfast (bread- water breakfast), or four hours after a 
test dinner (meat dinner). 

Fasting Stomach.- — If more than 10 c.c. is obtained when the 
stomach tube is passed in the morning there is retention and gross 
and microscopic food particles should be looked for. Titrate the 
acidity and examine for lactic acid according to the directions 
given under chemical examination after a bread-water breakfast. 

Bread-water Breakfast. — Give one shredded wheat biscuit 
and i pint of water or weak tea without sugar or cream and intro- 
duce the stomach tube at the end of one hour. With normal 
motility there is obtained less than ioo c.c. of odorless, colorless 
fluid containing particles of the carbohydrate food given. 

Bile gives a green or yellow color. Blood may be fresh, but 
more often is partially digested giving the "coffee-ground" 
appearance. Lactic, butyric and other organic acids give a sour 
odor. A putrid odor may result from necrotic stomach wall or 
from protein decomposition of retained food. An excess of mucus 
is shown by the tenaciousness of the material when the contents 
are poured onto the filter. 

Microscopic Examination (Fig. 10). — Filter into a ioo-c.c. 
graduate using a io-cm. funnel. Make a microscopic examination 
of the sediment on the filter. Starch granules show concentric 
laminations. Fat globules are highly refractive. Blood cor- 
puscles are not commonly seen as they rapidly disappear in the 
stomach contents. The nuclei of pus cells may persist after the 
cytoplasm has disappeared. Budding yeasts and yeasts in large 



GASTRIC JUICE 



19 



numbers are not normally present. Sarcines are abnormal and are 
most often found in cases of benign stasis. Oppler-Boas bacilli 
form lactic acid and are most often found in cases of malignant 
disease. 



£-' 




Fig. 10. — Gastric Contents. Microscopic findings, examine with high dry. 
a, Fat globules; b, red blood corpuscles; c, sarcines; d, starch granules; e, yeasts; /, 
Oppler-Boas bacilli. 

Chemical Examination. — One gram of the precipitate on the 
filter and 1 c.c. of the filtrate are placed together in a test-tube 
and examined for occult blood by the guaiac test (p. 24) ; the other 
chemical tests are performed on the filtrate. 

Giinzberg's Test for Free Hydrochloric Acid. — This acid is 
normally present in excess (so-called free hydrochloric acid) and 
the determination of its presence is important. Giinzberg's test 
is made by adding 5 drops of the filtrate to 5 drops of Giinz- 
berg's reagent (phloroglucin 2 gm., vanillin 1 gm. 7 and absolute 
alcohol 30 c.c. The reagent must not be older than two months) 
in a small porcelain dish which is evaporated to dryness on a 
water bath. A bright red color shows the presence of a mineral 
acid. 

As an additional test Congo paper may be placed in the filtrate. 



20 



HOSPITAL LABORATORY METHODS 



If free hydrochloric acid is present the paper is turned blue. 
Gunzberg's reagent reacts to a smaller amount of a mineral acid 
than is shown by this test. 

Phenolphthalein 



Alizarin 



Dimethyl 



FreeHCl + A. S. & O. A. +L. Comb. HC1 

Quantitative Titration of the Acids (Fig. n).- — Place 5 c. # c. 
gastric filtrate in a 30-c.c. Erlenmeyer flask (e), add 1 drop 
dimethylamidoazobenzol (J^ per cent, alcoholic solution) and run 
in decinormal NaOH until the bright red color is lost and a yellow- 
ish color appears. If there is no free HO, no red color is de- 
veloped when the indicator is added. If for example, 1 c.c. tenth- 
normal alkali is required to discharge the red color, then 1 times 20 or 
20 c.c. decinormal solution would be required to neutralize 100 c.c. 
of the filtrate using the dimethyl as an indicator. The amount 
of decinormal alkali required to neutralize 100 c.c of the gastric 
juice is taken as the numerical expression of the acidity. Thus in 
this case the free hydrochloric is 20. Normally it varies between 
about 20 and 40. Now add 1 drop phenolphthalein (^ per cent, 
alcoholic solution) and continue to run in the tenth-normal alkali 
until the first tinge of pink appears. The number of cubic centi- 
meters of alkali required to produce the pink times 20 gives the 
total acidity. This should lie between 40 and 70. 

To a second 5 c.c. of the filtrate add 1 drop sodium alizarin 
monosulphonate (^ per cent, aqueous solution) and run in the 
tenth-normal alkali until the first tinge of red appears. This 
indicator titrates the organic acids and acid salts in addition to 
the free HC1. The difference then between the alizarin titration 
and the dimethyl titration gives the organic acids and acid salts 
(A. S. &O.A.) (about 4). 

The estimation of the organic acids and acid salts by this 
method is of doubtful value. Lactic acid reacts to the dimethyl 



GASTRIC JUICE 



21 



and is the chief source of error. The difference between the total 
acidity and the alizarin titration gives the loosely combined 
hydrochloric acid (L. Comb. HC1). 




C>=cgS|) 



@ <m 
o 

a 



3 =Q 



c 



Fig. ii. — Gastric Contents. i\pparatus for gastric titration, a, Dimethyl in 
40-c.c. dropping bottle; b, alizarin; c, phenolphthalein; d, filter paper; e, 50-c.c. 
Erlenmeyer flask;/, pipette containing decinormal alkali. 



Lactic Acid (Kelling).- — Place 14 c.c. distilled water in a test- 
tube and add 1 c.c. gastric filtrate and 1 drop 10 per cent, ferric 
chloride. To a second tube of exactly the same size add 15 c.c. 
water and 1 drop 10 per cent, ferric chloride. If lactic acid is 



2 2 HOSPITAL LABORATORY METHODS 

present a canary-yellow color develops in the first tube. This is 
seen by looking straight into the mouth of the tube against a white 
background and comparing this tube with the one that contains 
ferric chloride alone. 

Pepsin. — Capillary glass tubes 2 mm. in diameter and 20 cm. 
long are filled with egg white by suction and the ends plugged 
with bread crumbs. Boil five minutes. Preserve in glycerine. 

Dilute 1 c.c. gastric contents in a 30-c.c. Erlenmeyer flask 
with 15 c.c. twentieth-normal HC1 and place in it 2 cm. of the 
albumin tube after washing off the glycerine. Place in the 
incubator for twenty-four hours. The number of millimeters 
digested from the two ends in this time may be recorded. 

Rennin. — Add 5 drops filtrate to 10 c.c. neutral milk and place 
in the incubator for fifteen minutes. Coagulation shows the pres- 
ence of rennin. 

Starch. — To 5 drops filtrate in a porcelain dish add 5 drops 
Lugol's iodine. A blue color indicates starch. 

Meat Dinner. — At noon give the patient clear broth, }i pound 
beefsteak, a small amount of mashed potato and one roll. 

Introduce the stomach tube in four hours. If the stomach has 
emptied itself, its motor power is good, and on the following day 
the stomach tube should be used one hour earlier after administra- 
tion of the same dinner. Undigested meat at the end of four hours 
shows deficient digestion. In a normal stomach there may 
remain on chemical examination only a small amount of almost 
completely digested food with a trace of free hydrochloric acid. 
The chemical examination is carried out just as in the case of the 
bread-water breakfast. 

FECES 

Introduction. — The fresh specimen should be sent to the labora- 
tory in a covered container and at once place in an incubator until 
examined. Undigested food particles, substances escaping from 
the biliary tract, occult blood, and parasites are the usual things 
examined for. 



FECES 



23 



Undigested Food Particles (Fig. 12).- — With a platinum loop 
place a small amount of the stool on the center of a slide, mix with 
water if necessary, apply a cover-glass and examine first with the 
low and then the high objectives. 

In faulty digestion muscle fibers are numerous and have dis- 
tinct striations and square (not^rounded) ends. Much fibrous 




Fig. 12. — Feces. Undigested food particles, a, Undigested muscle fiber; 
b, partially digested muscle fiber; c and d, vegetable cells; e, vegetable hair;/, vegetable 
spiral; g, fatty acid crystals; h, undigested connective tissue. 

connective tissue indicates faulty gastric digestion. Vegetable 
cells, spirals (the vessels of plants), and hairs are numerous in 
normal stools. 

Substances Escaping from the Biliary Tract. — To find gall- 
stones, the stool is tied up in a bag made of two thicknesses of 
surgical gauze, placed in a vessel and then washed under the tap. 
After washing out everything possible, cut the string with which 
the bag is tied, spread out the gauze with forceps and examine for 
stones. 

Bilirubin after escaping into the intestine is reduced by the 
bacteria present there to hydrobilirubin. To test for this pigment 



24 HOSPITAL LABORATORY METHODS 

rub up a gram mass in a mortar with 3 c.c. saturated aqueous solu- 
tion of bichloride of mercury and set aside twenty-four hours to 
dry. The particles that contain hydrobilirubin are red while 
those containing bilirubin are green. (Schmidt). 

Occult Blood (Guaiac Test) .—Place 1 gm. feces in the bottom 
of a test-tube with a glass rod, with a platinum loop, or, if fluid, 
with a pipette and add 5 c.c. glacial acetic acid and shake. Extract 
with ether by adding 20 c.c. ether to the tube and shaking gently, 
holding the thumb firmly over the mouth of the tube. 

Decant 5 c.c. of the clear supernatant ether extract into a 
second test-tube, add an equal amount of distilled water, 1 c.c. 
hydrogen peroxide, a knife-point of powdered guaiac and shake. 
A blue color in the ether is a positive test. A blue color is given 
not only by hemoglobin but also by red meats and green vege- 
tables (these should be eliminated from the diet), by pus, by the 
heavy metals and by certain other things less commonly. A 
negative test shows the absence of blood. 

Parasites. — Examination of stools for parasites is of great 
importance, especially in the tropics. The specimens for exami- 
nation should be obtained by giving a saline cathartic. 

Bacteria. — The bacterial content of the gastro-intestinal tract 
has frequently been made the subject of investigation but there 
are few organisms demonstrable in the intestine as the cause of 
specific lesions. 

The demonstration of tubercle bacilli is an indication of tuber- 
culous ulcers of the intestine only when it is known that the patient 
has not swallowed tuberculous material coughed up from the lungs. 
The anus must be thoroughly cleansed to eliminate the chance of 
error due to smegma bacilli present there. Tubercle bacilli are 
examined for by selecting suspicious looking masses of mucus and 
staining as in the case of sputum (p. 30). 

Dysentery bacilli and bacilli . of the typhoid-colon group are 
the commonest bacteria sought for identification. They are 
identified by isolating the organisms by means of successive streaks 
on plates of the proper media and agglutination of selected colonies 



FECES 25 

from the plates with known positive sera specific for the suspected 
organism. 

Ameba. — Entameba Histolytica (Fig. 13). — Choose blood- 
stained mucus, if present, from the fresh warm stool and place 
a small particle on a warm slide or warm stage and apply a cover. 
With the high dry lens this ameba has a clear ectosarc and in its 




Fig. 13. — Feces. Protozoa, a, Entameba histolytica — 1, ectoplasm; 2, endo- 
plasm; 3, nucleus; 4, cellular and bacterial inclusions; b, entameba coli; c, lamblia 
intestinalis with four pairs of nagella; e, trichomonas intestinalis with two pairs 
of nagella attached to the anterior end; /, cercomonas with a single nagellum at its 
anterior end. 



cytoplasm there are usually cells and bacteria. The nucleus is 
indistinct. This ameba is about five times the size of a red blood 
corpuscle. 

The entameba coli is a common inhabitant of the human 
intestinal tract. It is one to three times the size of a corpuscle, 
has a distinct nucleus and is only slightly phagocytic for corpuscles 
and other cells. It is not pathogenic. 

Flagellata. — Cercomonas hominis , trichomonas intestinalis, and 
lamblia intestinalis are flagellates found in the intestinal tract. 



26 



HOSPITAL LABORATORY METHODS 



They may be seen with the high dry objective. They have not 
been shown to be pathogenic. 

Infusoria. — Balantidium *coli is a pathogenic ciliate that 
produces ulceration of the colon. It may be found in the 
stools. 

Nematodes. — A large number of the nematodes are parasitic 
for man. Uncinaria americana and ankylostoma duodenale, the 




Fig. 14. — Feces. Ova. a, Unicaria americana or ankylostoma duodenale; 
b y strongyloides stercoralis; c, oxyuris vermicularis; d, tricocephalus dispar; e, 
ascaris lumbricoides; /, schistosoma hematobium; g, tenia saginata or solium; h, 
dibothriocephalus latus; i, tenia nana;/, hooklet from echinococcus cyst. 



new and old world forms of the hook-worm, are the most important 
of the nematodes (round-worms). The eggs are characteristic and 
offer the chief means for diagnosis (Fig. 14). Examine by placing 
a small amount of the liquid stool on the center of a slide, applying 
a cover and examining with the high dry lens. 



FECES 



27 



For a more searching examination place about one ounce of 
feces in a pint conical measuring glass, fill up with water and 
thoroughly mix with a glass rod. After sedimenting pour off the 
supernatant fluid and repeat. Finally examine the layer of finest 
sediment. 

The adult worm is present in the fresh stool only after treat- 
ment. Embryo worms may be obtained by placing ova-contain- 
ing stools in the incubator for a few days. The ova of the new 
and old world forms of hook-worm are not readily differentiated. 




Fig. 15. — Feces. Worms. Natural size. Examine first with low power. 
a, Ankylostoma duodenale; b, uncinaria americana; c, trichinella spiralis; d, trico- 
cephalus dispar; e, strongyloides stercoralis; /, oxyuris vermicularis; g, segment of 
tenia saginata; h, segment of dibothriocephalus; i, segment of tenia solium; /, 
tenia nana. 



Strongyloides stercoralis is diagnosed by finding the embryos 
(Fig. 15), as the eggs are not commonly found in the stool. Ex- 
amine with the high dry lens. 

Oxyuris vermicularis is the common pin-worm or seat-worm. 
Both eggs and adult worms may be found in the feces. The former 
are most often found in scrapings from about the anus and this 
is the most important means of diagnosis. Scrape off the material 



28 HOSPITAL LABORATORY METHODS 

with a dull scalpel, place on a slide with a very small amount of 
water and examine with the high dry lens. 

Tricocephalus dispar is the whip-worm and is diagnosed by 
finding the ova in the feces. 

Ascaris lumbricoides is the common round-worm. Both the 
adult worms and the eggs may be found in the stools. The latter 
may have ragged albuminous capsules or they may be naked. 

Trichinella spiralis is difficult to demonstrate in the feces. 
Place the feces obtained by active purgation in a tall cylinder and 
dilute with water. Pour the sediment into a large glass dish so 
as to form a thin layer and examine for minute worms against a 
black background. 

Trematodes. — Infections with trematodes or fluke-worms are 
not common in America and the cases found are importations. 

Schistosoma Hematobium. — The eggs of this parasite may be 
found in either the feces or urine. The adult worm lies in the 
mesenteric vein and in the veins of the bladder and vagina. The 
eggs penetrate through the walls of these vessels and reach the 
lumen of the bladder and of the intestine. 

Cestodes. — Some of the tapeworms are common intestinal 
parasites and are often diagnosed by the patient by his finding 
segments in the stools. 

Tenia saginata infections are diagnosed by finding segments 
by the method used for finding gall-stones or by finding the ova 
in the stools. The ova from the beef tapeworm will not infect 
man if ingested as the cysticercus bovis develops only in the 
beef. 

Tenia solium is differentiated from the saginata by finding 
the segments. The ova of the two are very similar. It is not 
common in this country. The eggs infect directly. Raw pork 
is the common source of infections. 

Dibothriocephalus latus, like the two preceding tapeworms, is 
large. Eggs and segments are both present in the stools. Fish 
is the source of infection. 

Tenia nana is the dwarf-worm. It is found in children and is 
diagnosed by finding the ova in the stools. 



SPUTUM 

SPUTUM 



29 



Introduction. — The saliva and the mucous secretions from the 
nose, mouth and throat, as well as the material coming from below 
the larynx, are often spoken of as sputum. For this reason it is 
important to learn from the patient, if possible, whether the speci- 
men was raised through the larynx by an expiratory effort. To 
obtain a specimen as free as possible from the nasal and oral 
secretions it is usually best to collect the material that is brought 
up through the larynx in the morning. 




Fig. 16. — Sputum. Paper sanitary cup and finger bowl. The cups are lined 
with heavy paraffined paper. These cups may be obtained from boards of health . 
The glass finger bowl measures 4.5 inches across the top. 

Gross Examination. — Sputum is now usually sent to the 
hospital laboratory in one of the many varieties of paper cups 
(Fig. 16) provided for this purpose. The specimen is emptied 
into a glass finger bowl (Fig. 16) and the empty cup filled with 
10 per cent, formalin. The sputum is then examined in the finger 
bowl against a black table top for gross structures. Pin-head 
to larger caseous masses of a yellow to white color may be found in 
tuberculous sputa. In non-tubercular cases of putrid bronchitis 
similar masses made up of masses of organisms and fat crystals 
may be found (Dittrich's plugs) (Fig. 18). Casts of bronchioles 
are found in pneumonic sputum as threads 2 mm. thick and a 
few centimeters long (Fig. 18). Casts of bronchi are found in 
fibrinous bronchitis. They often present a tree-like structure 



3° 



HOSPITAL LABORATORY METHODS 



measuring several inches (Fig. 17). Curschmann's spirals (Fig. 
1 8), sometimes found in bronchial asthma, are about the size of 
the casts of the bronchioles, but when placed under the microscope 
they show a distinct central spiral arrangement. After com- 
pleting the examination fill the dish with 10 per cent, formalin. 

Fresh Microscopic Preparations (Fig. 19).- — At the time of 
making the gross examination it is often desirable to examine 

certain of the gross particles under the 
microscope. To do this the desired 
material is removed from the dish with 
a platinum loop and transferred to a 
slide and a square cover slip applied. 
These fresh preparations are later 
dropped into the formalin solution. 
Red blood corpuscles (if from a fresh 
hemorrhage they are normal in appear- 
ance, are arranged in rouleaux), white 
cells (pus cells, eosinophiles or endo- 
thelial leucocytes), epithelial cells (the 
pavement variety usually from the 
nose and throat), and elastic fibrils (to 
test treat with 10 per cent. KOH) 
may be recognized. Charcot-Leyden 
(bronchial asthma), hematoidin, cholesterin and fatty acid crys- 
tals may also be identified. 

Stained Preparations. — Slides are best for making sputum 
preparations. An examination for tubercle bacilli is the most 
common examination asked for, but it is often required to examine 
for pneumococcus, bacillus influenzae, bacillus mucosus capsulatus 
and other organisms as well as for red blood corpuscles, pus cells, 
and other cells. 

Ziehl-Neelsen Stain. — Tubercle Bacilli. — With a platinum 
loop cover three-fourths of a slide with selected particles of the 
sputum. Warming the slide slightly over a flame facilitates the 
spreading out of a tenacious sputum. After drying the preparation 
apply the stain: (1) carbol-fuchsin for two minutes gently steaming; 




Fig. 17. — Sputum. 
cast. 



Bronchial 



SPUTUM 



31 




Fig. 18. — Sputum. Pathologic macroscopic findings. Examine with naked 
eye. Identify under low power of microscope, a, Curschmann's spiral; b, cast or 
plug from bronchiole; c, plug from bronchus; d, caseous mass. 




Fig. 19. — Sputum. Pathologic microscopic findings. Examine with high dry 
or oil in fresh and stained preparations, a, Neutrophils (pus cells) ; b, red blood 
corpuscles; c, endothelial leucocytes with blood pigment (heart-failure cells); d, 
eosinophiles; e, squamous epithelial cells from the upper air passages; /, round 
epithelial cells from the air sacs; g, Charcot-Leyden crystals; h, hematoidin crystal; 
if cholesterin;,/, fatty acid crystals; k, elastic fibrils. 



32 HOSPITAL LABORATORY METHODS 

(2) wash with water and then with Czaplewsky until thinner 
portions of smear are faint pink (about one-half minute); (3) 
wash with water and counterstain (one-half minute) withLoeffler's 
methylene blue; (4) wash with water, dry in air and examine with 
oil immersion. (For the preparation of the solutions, see p. 80) 

McJunkin's Polychrome Stain for Blood. — The cells and 
other bacteria are best stained by covering thin smears with the 
stain for one-half minute, and then diluting with 2 parts of dis- 
tilled water and staining five minutes. Wash with distilled water, 
dry and examine with oil. 

Eosinophiles are most often found in bronchial asthma. Endo- 
thelial leucocytes containing large amounts of hemosiderin (heart- 
failure cells), are often present in chronic valvular disease, espe- 
cially mitral stenosis. In these cases hemosiderin-containing 
leucocytes are found in the alveolar walls and in the bronchial 
lymph nodes as well as in the air sacs. 

Gram's stain may be necessary to aid in the differentiation of 
the bacteria found in sputum (see p. 79). 

BLOOD 

Introduction. — Apparatus for collecting blood for the estima- 
tion of hemoglobin, for the determination of the number of red 
blood corpuscles and white blood cells, and for making smears of 
the blood, is carried to the bedside of the patient. For examina- 
tion of patients in their homes this apparatus may be placed in the 
hemocytometer case (Fig. 20). Provide the pipettes with tips 
made from small soft rubber corks. Place in the case a straight 
surgical cutting needle attached to a cork inserted in a small vial 
containing 95 per cent, alcohol (one of the automatic blood stick- 
ers is convenient) and a second small vial containing Hayem's solu- 
tion. A Tallquist scale removed from a book and cover-glasses 
may be placed in the case cover. 

Hemoglobin. — The Sahli method is practical and fairly ac- 
curate, but for close determinations the Miescher method should 



BLOOD 



33 



be used. The Tallquist scale answers only for rough estimations 
above 70 per cent. 

Tallquist Scale Method. — At the bedside the lobe of an ear or 
the tip of a finger is wiped off with cotton moistened with alcohol, 
wiped dry with dry cotton and pricked with the sticker. The 




Fig. 20. — Blood. Counter case, a, Slide with counting-chamber; b, rubber 
cork covering tip ofjwhite pipette; c, soft rubber tubing; d, red pipette provided with 
rubber cork; e, cutting needle in 95 per cent, alcohol; g, Hayem's solution; h, J£ P er 
cent, acetic acid. 



blood must flow freely. The second small drop is allowed to 
touch the filter paper provided with the Tallquist scale, and as 
soon as the blood has been absorbed by the paper it is slipped 
beneath the Tallquist scale and carefully matched. If the hemo- 
globin is below 70 per cent, a more careful examination by another 
method should be made. 
3 



34 



HOSPITAL LABORATORY METHODS 



Sahli Hemometer Method (Fig. 21) — The tube (b) graduated 
from o to 140 is filled to 10 with decinormal hydrochloric acid. 
To the acid in the graduated tube, 20 c.mm. of blood, obtained by 
filling the pipette id) to the 20-c.mm. mark, are added. Shake 
and allow to stand exactly one minute. The pipette is rinsed by 
drawing the blood-acid solution into it. At the end of the minute 



M 



JL 



u 



n 



jl 



Fig. 21. — Blood. Sahli hemometer. a, Vial containing decinormal hydro- 
chloric acid; &, graduated tube; c, tube with standard solution; d, pipette. 



water is added until the color matches that in the standard tube 
(c) when the two are placed side by side. The number marking 
the upper surface of the fluid in the graduated tube is the per- 
centage of hemoglobin. 

The tube with the standard solution, contains blood, with a cor- 
puscle count of five million, 1 part, decinormal hydrochloric 10 
parts, distilled water 40 parts, and glycerine 50 parts. The solu- 
tion in this tube, should be protected from the light, carefully 



BLOOD 



35 



stoppered with a cork covered with paraffin, and renewed at the 
end of each year. 

To cleanse the pipette draw through it ^ P er cent, acetic 
acid, alcohol, ether and then air. This may be done by at- 
taching to a suction pump (Fig. 22) or by filling and then ex- 
pelling the solution by means of a thick rubber bulb. A horse 
hair may be used to remove any accidental dirt but a wire should 
never be used. Hairs from the tail of a horse should be kept in 




a' b' c d 



Fig. 22. — Blood. Suction apparatus for cleaning pipettes, a, Hayem's solu- 
tion; b, J/2 per cent, acetic acid; c, 95 per cent, alcohol; d, ether in 50-c.c. cork- 
stoppered bottles supported by strips of adhesive or better set into a frame made of 
thin galvanized iron; e, is thick-walled rubber tubing with a 2-mm. lumen into 
which the upper end of the pipette is inserted. 



95 per cent, alcohol for this purpose. Occasionally it may be 
necessary to fill the pipette with nitric acid and set aside for several 
hours in this acid to remove albumin from the interior of the 
pipette. 

Miescher Hemoglobinometer Method (Fig. 23). — The blood is 
drawn up to the % mark on the pipette (d) and then filled to the 
mark above the bulb with 0.1 per cent, sodium carbonate. Mix 
and blow out into the half of the cell 15 mm. deep (a), filling the 
other half with water. The half filled with the water is placed 
above the scale (e). Seal the cell with the glass cover (b) and then 



36 



HOSPITAL LABORATORY METHODS 



the metal cap (c) . The metal cap has a slit which should be placed 
at right angles to the partition dividing the cell. The instrument 
is now placed in a dark room before a candle and the red scale (e) 
is slid along until the two colors match when the reading on the 
scale is recorded. Make five such readings, add them together, 
and divide by 5. Now transfer the diluted blood by means of the 
pipette to one-half of the 12-mm. deep chamber which is similar 




Fig. 23. — Blood. Miescher hemoglobinometer. a, Cell 15 mm. deep; b, glass 
cover; c, metal cover with slit; d, pipette; e } color scale moved by the thumb 
screw g; /, light reflector; h, Fleischl modification of the instrument is made by 
Reichert of Vienna. 

to (a) and proceed to make five more readings. Add these five 
readings together, take the average and multiply by 1 %2- Add 
the two averages together and divide by 2. In cases of anemia, 
draw the blood up to 1 on the pipette thus obtaining a 1-200 
dilution instead of a 1-300. In this case divide the final average 
by 1.5. To get the percentage of hemoglobin, follow the direc- 
tions for the scale that comes with each instrument. Clean the 
pipette as indicated under the Sahli method. 



BLOOD 



37 



Dare Hemoglobinometer Method (Fig. 24). — The deter- 
mination of hemoglobin with Dare's hemoglobinometer rests on the 
comparison of a film of undiluted blood of definite thickness with 
standardized (a) colored-glass disk. The finger or ear is pricked 
and the slit of the blood chamber (#, w) when brought into contact 





Fig. 24. — 1, Dare hemoglobinometer; 2, blood chamber detached. 



with the blood immediately fills by capillarity. The excess of 
blood is removed and the blood chamber replaced in its slot or 
carrier with the white glass facing out. Light the candle and 
looking through the eyepiece (U) rotate (r) until the colors match. 
The hemoglobin is read directly from the scale. Many prefer to 
use this instrument as a routine in preference to the Sahli. 

Enumeration of the Red Blood Corpuscles (Zeiss Hemocy- 
tometer) (Fig. 11). — The pipette marked 101 above the bulb is 
used in counting the red blood corpuscles, and the one bearing the 
mark 11 is used in making the dilution for counting the leucocytes. 
These two pipettes are filled at the time the blood is taken for the 
hemoglobin estimation. 

The red corpuscle pipette is placed in a small drop of blood 
that has freely escaped onto the skin surface and gentle suction 
made, using the tongue to shut off a small space in the front part 



38 



HOSPITAL LABORATORY METHODS 



of the mouth for this purpose. The blood is drawn into the pipette 
until it reaches the 0.5 mark when the suction is stopped and im- 
mediately withdrawn from the drop of blood. If the column of 
blood reaches more than 1 mm. above the 0.5 then draw on up to 
0.6 for the dilution. If the column of blood only reaches about 
1 mm. above the 0.5 mark then the excess may be expelled by 

























































/. 


g 














/ 
































































7 
















































, 
















h 
















































_- 
















I 
































































m 
































































e 


h 

























































Fig. 25. — Blood. Counting chamber. A mechanical stage is necessary for 
blood work. Counting by strips facilitates the enumeration ofthe corpuscles. The 
entire ruled area 3 mm. square is divided into nine equal squares of 1 sq. mm. each. 

touching the tip of the pipette very lightly to the adjacent dry 
skin surface. As soon as the blood has been drawn to the 0.5 
mark, place the tip of the vial in Hayem's solution (mercuric chlor- 
ide 0.5 gm., sodium chloride 1.0 gm. ? sodium sulphate 5.0 gm. and 
distilled water 200 c.c.) and draw up to 101. Place the finger over 
the tip of the pipette, shake one minute and apply the rubber cap 



BLOOD 39 

in case the pipettes are to be transported to a laboratory some 
distance away; if the laboratory is at hand this is not necessary. 

To make the count shake again for one minute, force i or 2 
drops out of the pipette, and let a small drop escape on to the 
center of the counting-chamber island (Fig. 25) by touching it 
with the tip of the pipette. Apply the cover-glass and allow the 
corpuscles to settle. The diluted blood, should just cover the 
island and be free from bubbles of air. When viewed tangentially 
Newton's rings should appear between the cover and the slide 
about the moat. 

The ruled part of the island (a b c d) is 3 mm. square, while the 
central finely ruled area (efgh) is 1 mm. square. The distance 
between cover and upper surface of island is 0.1 mm. A side (ef) 
of the central finely ruled square is divided into 20 equal parts. 
A strip (i,j, k, I, or m) finely ruled and measuring Mo by 1 mm. 
may then be used as a basis for the count. Such a strip of course 
is made up of 20 small squares. At least ten such strips must 
be counted and it is better to count five in the first preparation, 
and five from a second preparation made by wiping the diluted 
blood from the island and replacing it from the pipette. One of 
these strips has of course a volume of Mo X Mo X 1 = Moo 
c.mm. and the dilution is 1-200. Therefore multiply the average 
number of red blood corpuscles in one strip by 200 X 200 = 40,000 
to get the number of corpuscles in 1 c.mm. 

Other methods of enumerating the corpuscles are used. All 
the numerous rulings of the counting chamber agree in having a 
central finely ruled area 1 mm. square. Each side of this central 
square is laid off into 20 equal parts, thus dividing it into 400 
small squares. The small squares of course are Ho mm. square. 

In a method extensively employed the small square (Mo X 
Mo mm.) is used as a basis. The depth of the blood between the 
island and the under surface of the cover is Mo mm.; therefore 
the volume of blood above each small square is Mo X Mo X Mo 
or Mooo c.mm. The dilution is 1-200. Count the number of 
corpuscles in 80 small squares and obtain the average in each 
square. Multiply the average by 800,000 (contents X dilution) 



40 HOSPITAL LABORATORY METHODS 

to get the number of corpuscles in i c.mm. of undiluted blood. 
Example: 480 corpuscles in 80 squares = 6 cells as an average. 
6 X 800,000 = 4,800,000 corpuscles in 1 c.mm. Since we divide 
by 80 and again multiply by 800,000, the number of corpuscles in 
80 squares may be counted and four ciphers annexed to this 
number. 

To cleanse the counting chamber thoroughly wipe off with a 
soft dry cloth. The pipette is cleansed as in the case of the Sahli 
pipette (p. 35). 

Enumeration of the White Blood Cells. — The blood is drawn 
into the white pipette to 0.5 and this diluted with J^ per cent, 
acetic acid (a stock bottle containing a crystal of thymol is kept on 
hand) to the n mark. Fill the counting chamber (Fig. 25) as in 
the case of the red blood corpuscles and count all the leucocytes 
in the 9 sq. mm. To obtain the number of leucocytes in 1 
c.mm. multiply the number in 1 sq. mm. by 200 (contents X 
dilution). 

To facilitate the counting, count all corpuscles or cells touch- 
ing left-hand and upper borders. Do not count any corpuscles 
that touch the lower or right-hand borders. For example, al- 
though a corpuscle lies entirely without the area being counted, 
but barely touches the upper border, it is enumerated. 

In counting the corpuscles use the high dry objective and in 
counting the white cells use the low dry. There should be four 
to six million red blood corpuscles per cubic millimeter while 
the leucocytes have a normal variation of from five to ten thousand. 
To obtain the color index divide the percentage of hemoglobin 
by the percentage of corpuscles (5,000,000 corpuscles = 100 per 
cent.). If the index is below one the individual corpuscle is poor 
in hemoglobin. 

Coagulation Time of the Blood (Bogg's Coagulometer) (Fig. 
26). — A small drop of blood is placed on the lower surface of the 
conical glass disk (a) and the disk inserted into a moist chamber 
(b) . Place under low power of the microscope and bring the drop 
into focus. When clotting has taken place gentle pressure on 
the rubber tube (c) after pinching off the end of the tubing moves 



BLOOD 



41 



masses of the corpuscles but they spring back as an elastic mass. 
The normal coagulation time is three to eight minutes. 




a.. 

b 



d 



m^ 



Fig. 26. — Blood. Coagulometer. a, Conical glass disk; b, moist chamber. 
Compress the rubber tubing at c after pinching off at d. 

Blood Films. — Cover-glasses (No. 1, 22 mm. square) are placed 
in concentrated sulphuric acid over night, when, after removing 
all acid by washing out under the tap, they are covered with 95 
per cent, alcohol and finally transferred" 
to chloroform. The covers are removed 
from the chloroform with forceps and 
wiped dry between two small boards 
covered with boiled muslin so as not to 
bring the fingers into contact with the 
covers. Place in box or glass dish with 
forceps. The removal of all fat is an ab- 
solute requirement if suitable films are to 

be obtained. FlG - 27.— Blood film by 

the cover- glass method. 

Touch one fat-free cover at it's center with thumb and forefinger 
to the ear so as to pick up a drop of blood gft£ ^V^td 
the size of a pin-head and apply a second b and the lower one at c 
cover to it so that the corners may be 

grasped (Fig. 27). As soon as the blood has spread out be- 
tween the covers pull them apart, being careful to keep the two 
parallel. If slides are used the best smears are secured by 
spreading a small drop on a fat-free slide as shown in Fig. 28. 
The cover-glass method is preferable. 




42 HOSPITAL LABORATORY METHODS 

Differential Counting of the Leucocytes.- — The films when 
dry are stained with a polychrome blood stain. The polychrome 
blood stains differ from the eosin-methylene blue stains in that 
the nuclei stain reddish while the cytoplasm tends to stain bluish. 
Many of the commercial polychrome blood stains give only the 
eosin-methylene blue staining. In the Mcjunkin's polychrome 
stain for blood (obtained from Bausch and Lomb Optical Co.), 
the reaction is accurately adjusted by titration and a definite 
polychrome staining is secured. 

€ 



T1.....-1 



Jk_ 



Fig. 28.— Blood. Blood film by the slide method. Place a drop of blood 
twice the size of a pin-head on a fat-free slide b at a point a. Place the end of a 
second slide c in it, and after the drop has spread along the entire surface of contact 
d, the film is made by pushing c in the direction shown by the arrow. 

Two drops of the stain are placed on the cover-glass held in 
suitable forceps (Fig. 29) for one-half minute so as to fix the film. 
The stain is then diluted with 4 drops of distilled water and the 
diluted stain allowed to act for five minutes. Wash with distilled 
water for a few seconds, dry and mount in colophonium-xylol. 
If the preparation has been made on a slide (Fig. 29), cover with 
8 drops of the stain for one-half minute, dilute with 16 drops of 
distilled water and stain five minutes. Wash, dry and examine 
with the oil immersion. 

The three varieties of leucocytes of the normal blood, lympho- 
blastic, myeloblastic, and endothelial are well differentiated after 
this technic. The classes to be tabulated in the differential count 



BLOOD 



43 



are lymphocytes (22), neutrophils (72), immature neutrophils 
(4), eosinophils (1.5), basophiles 0.25 and endothelial leucocytes 
0.25. There is doubtful value in attempting to divide the 
lymphocytes into large (young) and old (small). The numbers 
given indicate normal counts that may be obtained. In count- 
ing it is necessary to include all cells up to the very margin of the 
film regardless of the method used in making the film. This 
precaution is especially important, however, in the case of films 
made on slides. 





Fig. 29. — Blood. Staining of cover-glass and slide preparations, a, 3 by 1 
extra white, medium thickness, and fat-free slide supported by a small bottle to 
prevent the alcoholic stain from running over the sides; b, 22 mm. square, number 1, 
fat-free cover-glass; c, Novy cover-glass forceps, a, b and c may be obtained from 
Bausch and Lomb. 



Lymphoblastic Cells (A, Fig. 30). — These cells come from the 
cells of the germinal centers of lymph nodules and under certain 
conditions (lymphatic leukemia) the young form of cell may be 
found in the peripheral circulation. The cytoplasm and the 
nucleus of these young cells stain less intensely than the same 
structures in the older forms, but the cells are otherwise like the 
lymphocytes. Very rarely plasma cells may be^found in the 
peripheral blood. 

The very young lymphoblastic cells average smaller than young 
myeloblastic cells; the former do not give the oxydase reaction. 
This reaction is carried out as follows: (1) Fix in formalin; (2) 



44 



HOSPITAL LABORATORY METHODS 



A 



A 




o 



r 



s 



d 




A 



B 



C 



a 






D 






a 






• • # ..* 



Fig. 30. 



BLOOD 45 

place in*a i per cent, solution of alpha-naphthol in i per cent, 
potassium hydrate for live minutes; (3) transfer to 1 per cent, 
aqueous solution of dimethyl-^-phenylenediamin (Merck) for 
five minutes; (4) wash and mount in glycerine jelly. The myelo- 
blasts cells are bluish (Schultze). 

The identification of the lymphocyte depends 'largely on nega- 
tive properties. As in all blood work the stain used must be a 
good one and its staining characters known in detail. The 
nucleus, as in the identification of all cells, is of less importance 
than the cytoplasm. The cytoplasm of the lymphocyte is a 
robin's egg blue if much spread out, or a dark blue if the rim of 
cytoplasm about the nucleus is narrow. The cytoplasm is usually 
entirely homogeneous but often nodal points of a heavier blue are 



Fig. 30. — Blood. Normal leucocytes of the peripheral blood stream. These 
cells are stained with a polychrome blood stain and drawn on the same scale with 
the aid of a camera lucida. Some of the variations in size are unquestionably the 
result of the smearing out of the blood. 

A. Lymphocytes, a, Young lymphoblast drawn from a smear from a case of 
acute lymphatic leukemia. This cell is rarely found in normal blood, b, Lympho- 
cyte; c, older lymphocyte corresponding to the lymphoid cells of the tissue; d, plasma 
cell, rarely seen in the peripheral blood. This cell is reproduced from a smear from 
a case of chronic appendicitis, e, f, g, and h are lymphocytes that contain "azur" 
granules. These cells are large and their cytoplasm is not dense. In some cases 
at least -as in e, the apparent large size has resulted from the smearing out of the 
blood. 

B. Neutrophiles. a, Neutrophilic myelocyte drawn from a smear from a case 
of chronic myelogenous leukemia. This cell is not found in normal blood, b and c, 
Young (immature) neutrophiles. These cells have neutrophilic granules, and in 
cases where there is a neutrophilic leucocytosis the percentage of these varies greatly. 
At present it seems advisable to place them in a separate class, d, Neutrophile. 
The aging of b and c into d is seen not only in the blood of myelogenous leukemia but 
also in normal blood. 

C. Eosinophiles. a with its larger and less broken nucleus is a younger cell 
than b. The granules are very coarse and in the best preparations are not intensely 
stained. A stain that colors these granules an intense red is not a good polychrome 
stain. 

D. Basophiles. a is a younger cell than b. The coarse granules take a purplish 
heavy stain. 

E. Endothelial Leucocytes. In size and morphology, these cells may closely 
resemble the neutrophiles b and c, but the cytoplasm is entirely free from reddish 
granules and stains a heavier blue, b, c, and d have the non-granular basophilic 
cytoplasm in which the nodal points are very apparent. These cells are rather 
uncommon (below 0.25 per cent.) in the normal peripheral blood. They should be 
studied in preparations from cases of typhoid fever before an attempt is made to 
recognize them in normal blood. 

F. Blood Platelets. These are detached portions of megakaryocytes. Note the 
central purplish granular portion and the peripheral blue portion. 



46 HOSPITAL LABORATORY METHODS 

apparent, and if the preparation is grossly overstained these 
nodes may appear as reddish granules thickly peppered in. 

In the usual cover-glass and slide smears, from 30 to 80 per cent, 
of lymphocytes shows scattered rather coarse reddish granules 
usually under twelve in number. The cells showing the granules 
are usually large with an abundant light-staining cytoplasm. 

These granules in the cytoplasm of lymphocytes have usually 
been called "azur granules. " They stain like the nucleus and 
appear to be particles of nuclear material. A certain number of 
them at least are artefacts, produced by rubbing off small particles 
of nucleus in making the smears. This is shown by the increase 
in the percentage of lymphocytes showing these granules in very 
thin films of the same blood. It is widely stated that the lym- 
phocytes of lymph nodes do not show these granules, but prepara- 
tions made so thin that the lymphocytes are the same size that 
they are in the blood films, show lymphocytes with " azur granules. " 
These cells have the other characters of lymphocytes; they do not 
phagocyte in experimental animals in vivo and in the human 
blood they do not phagocyte in vitro. They have the para-nuclear 
granules described by Schridde. The larger of these lymphocytes 
with light-staining abundant cytoplasm and "azur granules" have 
by some been called "large mononuclears." As noted above this 
abundant light staining cytoplasm is due to the pressing out of 
the cells on the glass. 

Myeloblastic Cells (B, C, D, Fig. 30).— The young cells of this 
series found normally only in the bone marrow are microscopically 
much like the young lymphoblasts. They average somewhat 
larger in size, give the oxydase reaction and do not show the 
Schridde granules. However, one of three varieties of granules 
soon appears in the cytoplasm. A transition form between the 
youngest parent myeloblast of the bone marrow and the mature 
neutrophile, eosinophile and basophile of the blood is known as a 
neutrophilic, eosinophilic, or basophilic myelocyte. The recog- 
nition of the last two offers no difficulties. In fact the cytoplasm 
of the mature myeloblastic cells possesses so typical an appearance 
due to the presence of granules that there is no difficulty in classi- 
fying them. The development of these mature cells from non- 



BLOOD 47 

granular young cells may be so conclusively followed in the bone 
marrow that there is now no question about the origin in the adult 
of these cells. 

In general, immature or young cells owing to their partial 
differentiation are more difficult to identify. The young forms of 
the eosinophile and basophile are easily recognized by the large size 
and characteristic staining of their respective granules. The 
granules of the neutrophiles are finer and much less conspicuous 
than eosinophilic and basophilic granules. Thus in the young 
neutrophiles as would be supposed the granules are imperfectly 
developed and the demonstration of these requires the use of a 
stain with an accurately adjusted reaction. The nucleus of 
such young neutrophiles varies between the two extremes, the 
round nucleus of the neutrophilic myelocyte and the nucleus of 
the mature neutrophile, that is, the nucleus may be identical 
with the nucleus of the endothelial leucocyte and these two cells 
have been extensively confused, usually under the term "transi- 
tionals." To avoid such confusion neutrophile granules must be 
demonstrated in the young (immature) neutrophiles. 

The Endothelial Leucocytes (E, Fig. 30). — These cells arise 
from the endothelial cells of the blood- and lymph- vessels. These 
leucocytes as found in the blood stream average distinctly larger 
than the mature neutrophile. In certain pathologic processes, 
for example typhoid fever, these cells may be seen becoming free 
definitely within a blood capillary following a mitosis. The 
nucleus of these cells may be round, but it is more often indented 
or horseshoe in shape, that is, it resembles the nucleus of the im- 
mature neutrophile. The cytoplasm, however, is free from 
granules and this differentiates it with certainty from young 
neutrophiles. In properly stained specimens the identification 
of the young neutrophile rests entirely on the presence of neutro- 
phile granules. The cytoplasm of the endothelial leucocyte is 
blue and the blue modal points are very distinct. This gives 
the cytoplasm a richly staining appearance with nodal points. 
It does not have the homogeneous appearance of the lymphocyte. 
The cytoplasm of the young neutrophile has a tinge of blue that 
varies in degree with the age of the cell. The endothelial leucocyte 



48 HOSPITAL LABORATORY METHODS 

should be studied in smears from typhoid fever cases before at- 
tempting to identify it in normal blood. Counts recently made 
on eleven typhoid fever cases show an average of 2.4 per cent, of 
these cells. 

Red Blood Corpuscles. — They stain an orange or pink. Micro- 
cytes have a diameter of less than 7.5 microns, "normocytes measure 
7.5 microns while the macrocytes are abnormally large. Poikilo- 
cytes are irregularly shaped corpuscles. Stippling is a term applied 
to a basophilic granulation of the corpuscle. If due to lead, a 
reaction is obtained on treatment ' with an alkaline sulphide. 1 
Polychromatophilia is a bluish staining of the corpuscle. 

Normoblasts are nucleated reds, with a diameter of 7.5 microns, 
microblasts are small nucleated red cells, while megaloblasts are 
nucleated reds measuring above 15 microns. The nuclei of nu- 
cleated reds are dense, round and often eccentric and the cytoplasm 
stains an orange to a blue. 

Platelets (F, Fig. 30). — These elements, about one-third the 
diameter of a corpuscle but often elongated, are detached portions 
of megakarocyte pseudopoda. They are bluish with purplish 
granules toward the center when stained with a polychrome 
blood stain. There are about a half million per cubic millimeter, 
and perhaps the best method of estimating their number is by 
comparison with the corpuscles in properly stained smears on 
cover-glasses. 

Protozoa in Blood Films. — At the present time malarial para- 
sites are of first importance. The Mcjunkin's polychrome stain 
for blood is very satisfactory for the demonstration of these para- 
sites, the chromatin staining red. In order to stain the parasites 
heavily for the study of structure, the author's polychrome stain 
for protozoa should be used. 

Mcjunkin's Polychrome Stain for Protozoa (obtained from 
Bausch & Lomb Optical Co.). 2 - — This stain is readily prepared; 
1 gm. of methylene blue (Grubler's B. X.), 50 c.c. of decinormal 
(no factor) sodium carbonate and 50 c.c. of glycerine (Merck 
U. S. P.) are placed in a 500-c.c. beaker and heated at 87 to 8c) C. 

l See Jour. Med. Research, May, 1915, page 271. 
2 See Jour. A. M. A., Dec. 18, 1915, page 2164. 



BLOOD 



49 



for one hour, constantly stirring with a mechanical stirrer (Fig. 
31). The carbonate solution that has been made decinormal 
by titration with a standard acid using methyl red as an indicator 
must be run in accurately from a burette. The heating is best 
carried out by placing the beaker in a water-bath regulated at 
94 to 96°C. Remove the beaker from the water-bath and pour 
the polychrome solution while hot ino a 100-c.c. graduate. Rinse 
the beaker out with 5 c.c. of distilled water to remove any car- 
bonate and add this to the contents of the graduate. 



# -^ 




Fig. 31. — Blood. Mechanical Stirrer, a, Small water or electric centrifuge; 
b, axle carrying the head in which the centrifuge tubes are placed. This head is 
unscrewed from the top of the axle and the wooden pulley c screwed on. c has one 
deep groove and should have a shallower one also, d, burette stand; e, small 
cylinder of wood through which runs a short metal tube. The lower end of this 
metal tube carries the glass stirrer /, while the upper end carries the pulley g. 



Into a second graduate, measure sufficient methyl alcohol 
(Merck Reagent or Kahlbaum acetone free) to make 100 c.c. 
when added to the polychrome solution. Pour the alcohol into 
a 4-ounce bottle and add 0.75 gm. methylene blue (Griibler's 
B. X.) and 0.25 gm. eosin (Griibler's yellowish, water soluble). 
After complete solution of the methylene blue and the eosin is 
secured by shaking, pour the polychrome solution into the bottle. 
The total volume is now 100 c.c. If the solution is not complete 
place in the paraffin oven at 52°C. one hour or more. 

To use add 1 drop of the stain to 1 c.c. of distilled water and 
4 



5o 



HOSPITAL LABORATORY METHODS 



float the cover-glass preparation that has been fixed for ten 

minutes or more in equal parts' of alcohol and ether or in methyl 

alcohol, on the surface of the diluted stain for 

thirty to sixty minutes. Wash with distilled water 

for one to five minutes, air dry and mount in 

) V colophonium-xylol. 

^ Occasionally a sample of Griibler's yellowish, 

water-soluble eosin is encountered that cannot be 

used. If the stain is made with such an eosin, red 

blood corpuscles stain a blue that cannot be washed 

out. Once an eosin is found that is satisfactory it 

should be kept for this purpose. 

Mcjunkin's Polychrome Stain for ! Blood 

(obtained from Bausch & Lomb Optical Co.). 1 — 

To prepare Mcjunkin's polychrome stain for blood, 

add 250 c.c. of methyl alcohol (Merck reagent or 

Kahlbaum acetone free), 0.25 gm. of eosin 

(Griibler's yellowish, water soluble), and 2^ c.c. of 

half -normal hydrochloric acid to 50 c.c. of the 

polychrome stain for protozoa. The half-normal 

acid is added accurately from a burette. Apply 

according to the directions for the differential 

staining of leucocytes (p. 42). 

Widal Reaction. — A Wright pipette (Fig. 32) is 

Fig. 32.— filled with blood from the patient's ear and the 
Blood. Wright r 

pipette. Fill serum allowed to separate out. One part of this 

curved C end g a to serum an d 29 parts of normal saline measured in a 
a large drop of capillary pipette are mixed in the well of a concave 
in a flame. After slide. One part of this dilution and one part of a 
ma^^scrVh^d twelve-hour bouillon culture of typhoid bacilli are 
at b with a file, mixed and a hanging drop preparation made from the 
serum removed mixture. If positive there should be agglutination 
with a capillary [ n one hour. A control hanging drop without the 
serum is always made. 
Phagocytosis in Vitro.- — To determine the ability of leuco- 




pipette. 



x See Jour. A. M. A., Dec. 18, 1915, page 2164. 



BLOOD 51 

cytes to incorporate foreign bodies in their cytoplasm, it is neces- 
sary to prepare in a prescribed way the three ingredients required 
in the reaction. 

Leucocytes. — First the leucocytes are prepared by placing 
4 c.c. 2 per cent, sodium citrate in a 15-c.c. graduated centrifuge 
tube and adding 1 c.c. blood from a finger or ear. Shake, make 
up to 15 c.c. with normal saline and centrifuge. Pipette off the 
clear supernatant fluid, again add normal saline to 15 c.c. and 
centrifuge. Pipette off again, add saline to 1 c.c. and shake. 
This is the preparation of leucocytes. 

Serum. — To enable leucocytes to take up actively foreign 
particles, the presence of fresh unheated serum is required. The 
substances present in the serum that enable the leucocytes to act 
in this way have been named opsonins. At the time the 1 c.c. of 
blood is taken from the finger or ear for the leucocytes a small 
Wright pipette is filled with blood which is allowed to clot. One 
end of the pipette is then sealed, the other end is broken off and 
with a minute glass thread, the clot is stirred up. This tube is 
placed in the centrifuge along with the preparation of leucocytes. 

Suspension of the Foreign Body. — This is usually a suspension 
of bacteria. Bacteria such as staphylococcus, streptococcus, or 
typhoid bacilli are grown for eighteen hours on an agar slant, the 
tube is half filled with normal saline and the growth rubbed off 
into the saline with a platinum loop. The suspension is pipetted 
off into another cork-stoppered tube and shaken in a mechanical 
shaker for an hour. 

To make the test thoroughly mix in the well of a slide equal 
parts of the leucocytes, serum and bacterial suspension. Use a 
capillary pipette for measuring. Place the pipette containing 
this mixture at 37°C. for fifteen minutes. Make smears by the 
slide method. Stain with a polychrome blood stain and count the 
number of bacteria in 100 leucocytes. The average number of 
bacteria per leucocyte is the phagocytic index of the individual 
whose serum is used. The phagocytic index of a patient divided 
by the phagocytic index of a normal (control) individual is the 
so-called opsonic index. 



52 



HOSPITAL LABORATORY METHODS 



Blood Cultures. — To make a blood culture carefully, not only 
is the inoculation of media in tubes and flasks usually required, 
but also the plating of the blood. As a result this part of the 



/ 



Q 



1 
8 — & 



-A 



aL 



Fig. 33. — Blood. Mcjunkin's blood tube, a, Oxalate solution; b, extra 
large test-tube; c, 3-mm. soft rubber tubing; d, cotton; e, small test-tube; 
/, large needle provided with a large stilette; g, cotton; h, 10-c.c. pipette; i, small 
test-tube. 



routine of a hospital laboratory is time consuming and laborious, 
chiefly for the reason that much apparatus must be carried to the 
bedside, where the inoculations are always made under certain 
difficulties. 

In the collection of blood for Wassermann tests, in which per- 



BLOOD 



53 



feet asepsis is not obligatory, a needle is inserted into the arm 
vein and the blood run directly into a sterile tube. It is the ease 
and speed with which a large number of specimens of blood may 
be collected for this test that prompted the use of a tube with 
oxalate solution in it and a needle attached. 

McJunkin's Blood Tube 1 (Fig. 33). — The preparation of the 
tube is simple. In an extra large test-tube there are placed 15 c.c. 
of a solution (a) which contains 2 gm. 
of ammonium oxalate and 6 gm. of 
sodium chloride to a liter of distilled 
water. Cotton 4 cm. w T ide (d) is now 
wrapped around the rubber tubing 
(c), which is 150 mm. long with a 
1 -mm. wall and 3 -mm. lumen, and 
into the upper end of this tubing there 
is inserted a 19-gage needle 1 inch long 
(/). The needle is capped by insert- 
ing into the upper end of the extra 
large tube a smaller one (g) about the 
lower end of which is wrapped a 3-cm. 
plug of cotton. Both cotton plugs 
should fit snugly. The tube com- 
plete is autoclaved for twenty minutes 
at no°C. 

After the needle has been inserted 
into the vein, a few seconds are re- 
quired for the blood to pass through 
the tubing. The lower cotton plug 
eliminates all chances of contamination and when it is removed 
in the laboratory, the upper end of the tube is flamed and the 
diluted blood transferred with a 10-c.c. sterile pipette (h) to the 
media that are indicated. The tubing and needle are cleansed 
by forcing through them a few cubic centimeters of water from a 
syringe, after which they are placed in a quart Mason jar in a 
saturated solution of borax. _ 

1 Jour. A. M. A., Mar. 7, 1914, page 774. 




Fig. 34. — Blood. Erlen- 
meyer flask for defibrinating 
blood. 125-c.c. sterile flask 
containing a dozen bits of fine 
mesh, well-galvanized wire gauze. 
Shake by hand or place in de- 
fibrinator, Fig. 35. 



54 



HOSPITAL LABORATORY METHODS 



Inoculation of Media. — In suspected cases of typhoid a flask 
of bouillon (p. 75) and a flask of lactose bile are inoculated with 
several cubic centimeters of blood. On the following day and 
each succeeding day, a transplant is made from each of the flasks 
to an- agar slant and a tube of litmus milk. If the agar slants 
show a growth of Gram negative bacilli and the milk is not changed, 
an agglutination test with a known typhoid serum is made. 

To obtain the agglutinating serum a rabbit is given five 
intraperitoneal injections of the typhoid bacilli at three- or four- 



...-■/ 



,h S 





mi Ljy 




J^ 6 \ 


\__^ r f y S ■'*■ D ■■>■■ ?,/ 

»I1_ / 




&""K^ 


F\ 


rr 


=^] 






; : 'M % 



Fig. 35. — Blood. Mechanical defibrinater and shaker (Barta). a, Heavy 
wooden block to be nailed onto platform at desired place; d is a 3^~ m cn 
iron rod firmly driven into block and supporting wooden pulley, b, and eccentric 
support, c. b is run from pulley shown in Fig. 29 by a cord; / is a J^-inch iron rod 
attached to the wooden pulley eccentrically and running through the T-shaped 
tube e; g, j and h are thin sheet iron; g and j are attached firmly. Tubes to be 
shaken are clamped into g while flasks are clamped intoj. 

day intervals. On the first day the animal is given a suspension 
of one-half an agar-slant culture. To prepare this suspension 
10 c.c. of sterile saline is added to the slant, the growth rubbed off 
with a platinum loop and the tube placed in water at 6o°C. for 
one hour. At the second and third injection give one tube and 
at the fourth and fifth injections two tubes. Bleed from the 
carotid ten days after the last injection. Agglutinating sera 
other than typhoid (dysentery and the paratyphoids especially) 
should be kept on hand. 



BLOOD 



55 



In cases other than typhoid, three glucose agar plates are 
poured. One flask of bouillon, one tube of lactose bile and one 
tube of litmus milk are inoculated as a routine. Other media 
are inoculated if indicated. Each day for at least five days the 
liquid media are examined and transplants are made to agar slants. 
The plates are examined daily and are kept for at least ten days. 
Transplants are made to the various special media as indicated. 




Fig. 36. — Blood. Bulb pipette. Secure soft glass tubing for blowing with 
8 mm. outside and 4 mm. inside diameter. Cut into 30-cm. pieces and constrict 
both ends a, and plug with cotton. Dry-heat sterilize. Heat the central portion 
and draw apart into two pipettes. Blow bulbs of desired size and then bend the 
pipette at a convenient angle. Such pipettes are used for transferring sterile fluids. 

With the usual technic, contaminations are rather common. 
For this reason the examiner must consider all possible data so 
as to determine whether the organism present came from the 
patient's blood. If there is no growth of pathogenic organisms 
on any of the media at the end of ten days the culture is reported 
as negative. 

Wassermann Test. — Introduction. — This test is based on 
the complement fixation reaction. The antigen is not specific and 
the reaction is not absolutely * specific for syphilis. Unless the 
clinician is thoroughly familiar with the examiner's technic it 
is advisable to provide him with the data contained in this and the 
following paragraph. A positive reaction not due to syphilis 
may be obtained in yaws, trypanosomiasis, and leprosy and some 



56 



HOSPITAL LABORATORY METHODS 



have observed it rarely in scarlet fever, malaria, lobar pneumonia, 
diabetes mellitus and immediately after ether anesthesia. A 
negative reaction in cases of syphilis may be obtained during treat- 
ment with salvarsan or mercury and is said to occur during acute 
alcoholism. 

In negative reactions all the corpuscles are hemolyzed, while 
in perfect positive ones there is no hemolysis. Between these 
^-v two extremes, all degrees of hemolysis 

occur. Those that show only a slight 
clouding are marked " positive +," which 
represents less than 10 per cent, inhibi- 
tion of hemolysis. Such a result has no 
diagnostic value except in cases of known 
syphilis under treatment. In such cases 
further active treatment is indicated. A 
greater clouding, but one in which there 
is distinct hemolysis in the clear liquid 
above the sedimenting corpuscles, repre- 
sents an inhibition of hemolysis varying 
between 10 per cent, and 90 per cent., 
" positive ++-•" Such a result should be 
considered as a positive diagnosis of 
d syphilis only in connection with strong 
clinical evidence. It calls for further 
active treatment. In cases where there 
is no tinge of red in the liquid above the 
sedimenting corpuscles, there is complete 
inhibition of hemolysis, "positive + + + •" 
Such a result is to be considered as a 
positive diagnosis of syphilis, if the con- 
ditions mentioned above are not present. 

Sera. — Blood is drawn from the vein at the bend of the elbow 
by means of a syringe and placed in sterile cork-stoppered test- 
tubes (120 X 13 mm.). The tube, with the brown slip wrapped 
about it, is placed on ice and the test made within forty-eight hours. 
Not less than 2 c.c. of blood should be collected and at times this 




Fig. 37. — Blood. Siphon 
pipette (Barta). A broken 
burette, a, is attached to the 
glass tubing, c, by means of 
rubber tubing, b. The fluid 
to be siphoned is pushed up 
on c as it is drawn off into a 
tumbler, d. 



BLOOD 



57 



amount may be collected from the ear and in other ways. When 
the test is to be made, the tube and its brown slip are given a 
number. The serum is removed from the tube with a capillary 
pipette made from glass tubing provided with a nipple for 
suction (use a different pipette for each serum) and paced in 
the same size tube bearing the same number. 

If the serum is not clear, then the clot in the original tube is 
thoroughly broken up with a sterile glass rod and the tube placed 





Fig. 38. — Blood. Rack for Wassermann tubes. 30 by 12 by 8 cm. made 
from thin galvanized iron. The ends are made in two pieces and crimped together 
at their center. This is not shown in the figure. 



in the centrifuge and whirled five minutes when the clear serum 
may be pipetted off. Use separate pipettes and glass rods for 
each specimen. After using, the rods, pipettes and dirty tubes'are 
placed in a wash basin and washed, after which they are dried and 
sterilized. The properly numbered tubes containing the clear serum 
are inactivated in a water bath at 55°C. for thirty minutes; 
0.1 c.c. of clear serum is used for each test. A i-c.c. pipette 
graduated in hundredths to the very tip is used for measuring 
the serum. Before measuring the second serum, the pipette is 
rinsed four times with sterile saline by drawing the solution into 
it and then blowing it out again. 1 c.c. of non-inactivated cere- 
brospinal fluid per test is used. By placing 0.1 c.c. of the serum 
in drops on paper and letting dry, a " positive + + +" serum 
may be kept for three months. 



58 HOSPITAL LABORATORY METHODS 

Complement. — If the number of reactions to be done is above 
thirty, it is perhaps better to obtain the blood by cutting the 
throats of guinea-pigs with a brain knife after a numbing knock 
on the head and allowing the blood to drip into a io-cm. porcelain 
dish. A second person defibrinates by stirring with a heavy 
nickel-plated wire or a heavy platinum wire. 

If there are fewer tests the blood is removed from the heart of 
the etherized guinea-pig strapped to a holder (Fig. 51). In this 
way several (8 c.c.) of blood maybe removed from a large pig. 
A fine platinum-iridium needle should be used. The blood is at 
once forced from the syringe into a sterile 30-c.c. Erlenmeyer 
flask containing six pieces (1 cm. square) of bright fine-mesh wire 
gauze (Fig. 34) . The flask is shaken by hand or in a mechan- 
ical defibrinator (Fig. 35). 

After the blood is obtained in either of these ways and defib- 
rinated, it is placed in sterile 15-c.c. centrifuge tubes and whirled 
for five minutes. Remove from the centrifuge and pipette off 
the clear serum. The same pipette that is used for measuring 
the serum maybe used for measuring the complement. 0.05 c.c. 
is used for each test. 

Amboceptor. — At intervals of three or four days (Tuesday 
and Friday of each week) inject 5, 8, 12, 15 and 20 c.c. of sterile, 
washed, sheep corpuscles into the peritoneal cavity of a rabbit 
weighing above 2000 gm. 

Another good method for obtaining amboceptor is to give four 
injections of one to three cubic centimeters intravenously and 
bleed ten days after the last injection. 

To obtain, the sterile, washed corpuscles, place 20 c.c. of de- 
fibrinated sheep's blood in a sterile 50-c.c. centrifuge tube and 
mark the upper surface of the blood with a blue pencil. Fill the 
tube with sterile saline and whirl for five minutes. Siphon off the 
supernatant fluid (Fig. 37), add saline and again centrifuge. 
Wash the corpuscle in this way four times. After decanting the 
saline the last time, add a sufficient amount of saline to bring the 
corpuscles to the blue mark. 

Ten days after the injection of the last dose, take sufficient 



BLOOD 59 

blood from the ear to test the strength of the serum according to 
the directions given under "Titration for Amboceptor Unit." 
If satisfactory, fasten the animal in a rabbit holder, shave the 
neck, anesthetize, and dissect out both carotids for at least one 
inch. Tie one carotid as high as possible and place a spring artery 
clamp below. Cut just below the ligature, secure a firm hold 
in the adventitia of the loose end of the artery so as to guide the 
vessel into the mouth of the flask and then remove the clamp. 
The blood is collected in a sterile 250-c.c. Erlenmeyer flask. 






g 



a b c d e f 

Fig. 39. — Blood. Block for amboceptor, etc. a, 50-c.c. centrifuge tub con- 
taining saline for washing pipettes; b, 50-c.c. centrifuge tube containing saline for 
making dilutions; c, 50-c.c. centrifuge tube containing 5 per cent, suspension of 
washebl sheep's corpuscles; d, 155 by 16 mm. test-tube for antigen-complement — 
ambocep tor-saline mixture of which 1 c.c. per test is used; e, properly diluted antigen; 
/, properly diluted amboceptor; g, block of wood 12 by 2^ by ij^ inches. Each 
tube is provided with its own pipette before beginning the tests. 

Place on ice and allow the clot to harden over night. Remove 
and inactivate the serum. Seal 0.1 c.c. in small Wright pipettes 
(Fig. 32). Make the proper dilution (1-100 or 1-200) just before 
making the tests. Measure with the i-c.c. pipette graduated in 
hundredths. For the test use two units. 

To determine one unit, dilute the inactivated serum 1-100 or 
1-200 and use the amounts indicated under " Titration for Ambo- 
ceptor Unit." The smallest amount that completely hemolyzes 
all the corpuscles is one unit. It should be less than 0.07 c.c. of 
the 1-100 dilution. If the serum is not stronger than this at the 
time the test titration is made on the ear, it is preferable to 
immunize another animal. 

Antigen. — The antigens used in the Wassermann test are not 
specific and more changes have been made in the antigens since 



60 HOSPITAL LABORATORY METHODS 

the test was first described than in any other part of the technic. 
At the present time the cholesterin antigen is said to give positive 
reactions in syphilitics when the acetone-insoluble antigen gives 
negative results. On the other the cholesterin antigen appears 
to give a greater number of positive reactions in non-syphilitics. 
Again no serum gives a positive reaction with acetone-insoluble 
antigen and a negative one with cholesterin. 

For these reasons all the sera are tested with the cholesterin 
antigen first and if negative no further tests are required. The 
sera that are positive with cholesterin antigen are done with the 
acetone-insoluble antigen and the results with both antigens 
reported. The significance of "positive +," "positive + + ," 
and "positive + + + " given for the interpretation of results 
in the introduction refers to the acetone-insoluble antigen. The 
result with the acetone-insoluble antigen is the one that should be 
considered in the initial diagnosis of a case, while the result with 
the cholesterin antigen is of value in cases under treatment. 

Cholesterin Antigen. — To 500 c.c. of absolute alcohol, add 
2 gm. of cholesterin (Kahlbaum). Before making the test titra- 
tions, add 9 c.c. of saline to 1 c.c. of this alcoholic solution. After 
the titration make such a dilution that 0.1 c.c. of it is used per 
test. That is dilute so that 0.1 c.c. = twice the antigenic dose. 

With the cholesterin antigen some use for each test four 
instead of two times the antigenic dose. This is possible, for the 
anticomplementary property of the cholesterin antigen is much 
less than that of the acetone-insoluble. The i-c.c. pipette 
graduated in hundredths may be used. For the test use twice the 
antigenic dose. 

Acetone -insoluble Antigen. — Place in a pint Mason jar 50 
gm. fresh human or beef heart that has been ground in a sausage 
grinder and add 450 c.c. absolute alcohol. Place in the incubator 
at 37 for ten days shaking repeatedly. Filter through paper, 
place the filtrate in a flat dish 12 inches across and evaporate 
with an electric fan. Take up the residue in a dish with 200 c.c. 
of ether. Place the milky ether in a Mason jar over night. The 
following morning decant the ether which is now clear into a 



BLOOD 6l 

beaker and evaporate to about 50 c.c. Then add 450 c.c. of 
acetone (C. P.) to the 50 c.c. remaining, thoroughly mix and 
decant the supernatant solution from the insoluble residue. 
After decanting, it is usually necessary to evaporate the acetone 
extract to the desired sticky mass with a fan. The sticky mass 
contains the antigenic lipoids. To 0.3 gm. add 1 c.c. ether and 
9 c.c. of methyl alcohol. 

To 1 c.c. of this ether-alcohol solution add 9 c.c. of saline and 
make the test titrations as outlined below. If the titration is 
satisfactory seal in tubes about 2 c.c. of the ether-alcohol solution 
per tube. Each time before starting to make Wassermann tests, 
such a dilution is made with saline that 0.1 c.c. of the resulting 
emulsion contains twice the antigenic dose. The stock antigen 
in the 2-c.c. tubes keeps for several years. 

Gonococcus Antigen. — Grow the gonococci twenty-four hours 
on hydrocele or ascitic agar slants, take up the growth from each 
tube with 2 c.c. of distilled water by means of a bulb pipette 
(Fig. 36) and place the suspension of gonococci in a 500-c.c. glass- 
stoppered bottle. Shake the bottle in a mechanical shaker (Fig. 
35) over night. Heat at 56°C. for two hours. Filter through a 
Berkefeld filter and add 0.9 per cent, sodium chloride and 0.5 
per cent, phenol. Make such a dilution of this solution that 
0.1 c.c. contains twice the antigenic dose. Some prefer to make 
gonococcus antigen from a number of different strains of the 
organism. Bacterial antigens other than gonococcus are prepared 
in the same way. 

Testing the Antigens. — All antigens must be tested out be- 
fore using. To make these initial tests, 9 c.c. of saline are added 
to 1 c.c. of the stock antigen. In order that 0.1 c.c. contain the 
amount that must be used for each test, variations from this 10 
per cent, emulsion are later made according to the result of these 
titrations. 

Hemolytic Property. — If an antigen is hemolytic in ten times 
the antigenic dose it should not be used. To test place 1 c.c. of 
the 10 per cent, emulsion, 1 c.c. of saline and 0.5 c.c. of 5 per cent, 
suspension of corpuscles in a test-tube and incubate one hour at 



62 HOSPITAL LABORATORY METHODS 

37.5°C. The liquid above the sedimenting corpuscles should not 
be tinged with red. 

Anticomplementary Property. — Ten times the antigenic dose 
should not be anticomplementary (i.e., it should not contain a 
substance that destroys the action of the complement and thus 
gives false positives). To test, place i c.c. 10 per cent, emulsion 
of antigen, i c.c. saline, 0.05 c.c. complement and two units ambo- 
ceptor in a test-tube and incubate thirty-five minutes at 37.5°C. 
Then add 0.5 c.c. of 5 per cent, suspension of corpuscles and place 
in the incubator at 37.5°C. for one hour. 

It is usually -preferable, however, to determine the exact 
amount that is anticomplementary. To do this, instead of setting 
up a single tube as above, place eight tubes in a rack and add a 
decreasing amount of the antigen to each: 2, 1.5, 1, 0.8, 0.6, 0.4, 
0.2, and 0.1 c.c. of the 10 per cent, emulsion. The other substances 
are added in the amounts indicated above in the case of the single 
tube where 1 c.c. of antigen is used. The tube containing ten 
times the antigenic dose as determined below should show complete 
hemolysis. 

Antigenic Property. — To determine the antigenic property of a 
new antigen run the titration according to directions for the 
actual test (see p. 65), except that eight tubes are set up 
instead of one, and in these tubes there are placed decreasing 
amounts of the emulsion of antigen: 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 
0.05 and 0.02 c.c. The serum added to each tube is 0.1 c.c. of a 
known " positive + + + " serum. 

Such dilutions of the stock antigens are made that 0.1 c.c. of 
these will be twice the smallest amount that gives complete in- 
hibition of hemolysis in the test of the antigenic property. Ten 
times this proper antigenic dose should not be hemolytic and 
should not be anticomplementary. 

In the case of cholesterin antigen, there is no difficulty in 
obtaining an antigen that fulfills these requirements, but it is 
much more difficult to secure an acetone-insoluble antigen that 
is not anticomplementary in ten times the antigenic dose. In the 
case of bacterial antigens it is even more difficult, and in some 



BLOOD 63 

cases it may become necessary to use an antigen that becomes 
anticomplementary at three times the antigenic dose. This is 
the very shortest " working distance' 7 that is permissible. 

Corpuscles. — Collect the blood from slaughter-house lambs in 
a sterile pint Mason jar containing glass beads. Collect half full 
and then shake until completely defibrinated (ten minutes). 
Keep on ice until used. With a 10-c.c. pipette transfer 10 c.c. 
of the defibrinated blood to a 50-c.c. centrifuge tube and mark the 
upper limit of the blood with a blue pencil. Make up to the 
50-c.c. mark with the saline and then centrifuge 5 minutes. Re- 
peat this three times. After the last centrifugation make up to 
the original 10 c.c. (blue mark) and to these 10 c.c. add 190 c.c. 
of saline in a 250-c.c. Erlenmeyer flask. Keep on ice. Use a 
10-c.c. pipette graduated in tenths for measuring out the cor- 
puscles. For the test use 0.5 c.c. of the 5 per cent, suspension of 
corpuscles. 

For a limited number of Wassermann tests, it is often pref- 
erable to remove the blood from the jugular vein of a sheep kept 
for this purpose. One person should straddle the sheep holding 
its chin up while the other clips off the wool and inserts into the 
vein a small platinum-iridium needle attached to a 20-c.c. syringe. 
Defibrinate the blood in a 250-c.c. flask (Fig. 34) containing disks 
of wire gauze. Wash in 15-c.c. centrifuge tubes. A siphon 
pipette (Fig. 37) is very convenient for removing the supernatant 
fluid while washing. Never vary the technic of making up the 
corpuscle suspension. 

Saline. — Add 8.8 gm. sodium chloride (Merck U. S. P.) to a 
liter of distilled water. Autoclave. For use place the saline in 
50-c.c. centrifuge tubes supported in a block (Fig. 39) in which 
there are placed 10-c.c. pipettes graduated in tenths. There 
should be two of these; one is used for washing the pipette used 
for measuring the sera and the other for diluting the ingredients 
to the correct amounts. 

Test. — Minor modifications of technic are very numerous. 
However, the general principles of the test are the same in all 
laboratories where the most pains-taking care is used. In a test 



64 HOSPITAL LABORATORY METHODS 

where the technic is so complicated, there is no excuse for 
introducing a factor which will certainly increase the chance of 
error. 

In the test, six liquids are placed together in a single test-tube: 
patient's serum (o.i ex.), complement (0.05 c.c.), amboceptor 
(two units), antigen (0.1 c.c. = twice the antigenic dose), cor- 
puscles (0.5 c.c), saline (q.s. 1.6 c.c). The patient's serum and 
the corpuscles are added separately but the use of 1 c.c. containing 
the other four (an antigen-complement-amboceptor-saline mix- 
ture) reduces the number of measurements for each test to 
three. 

Before beginning the test (this may be done while the " Titra- 
tion for amboceptor unit" is being made), fill tube e, Fig. 39, with 
antigen. Suppose in the titration of the antigen for antigenic 
property it is found that 0.04 c.c. of a trial 10 per cent, emulsion 
is the antigenic dose. Then 0.08 c.c. of a 10 per cent, emulsion 
or 0.1 c.c. of 8 per cent, emulsion is twice the antigenic dose. 
In this case in order that 0.1 c.c. may contain twice the antigenic 
dose dilute the stock antigen 8-100. The dilution that is to be 
made is marked on each stock antigen. 

As soon as the " Titration for amboceptor unit" is complete, 
fill tube d, Fig. 39. This is done according to " antigen-comple- 
ment-amboceptor-saline table." 

The sera to be tested are inactivated, numbered consecutively 
and placed in the front row of a rack (Fig. 38). 

The 5 per cent, suspension of corpuscles is in c, Fig. 39. The 
three liquids then to be used in the actual test are in the test-tube 
rack, Fig. 38, and in c and d, Fig. 39. 

Titration for Amboceptor Unit. — Place eight test-tubes (120 X 
13 mm.) in the front row of a rack (Fig. 38) and to each of these 
add the amounts of diluted amboceptor (Fig. 39) indicated below. 
Add to each tube 0.05 c.c. 5 per cent, suspension sheep corpuscles 
and 1.0 c.c. saline. 

Incubate in the water-bath thirty-five minutes at 37-5°C. 
The last tube in which hemolysis is complete is one unit. This 
should be between 0.02 and 0.07 c.c. 



BLOOD 



65 



Amboceptor diluted 


. . . 1 0.08 0.07 0.06 0.05 


0.04 0.03 0.02 O.OI 


Complement undiluted G. 
serum 


... O.Os O. Os 0.0^ 


0.05 


0.05 


0.05 


0.05 


0.05 




Sheep corp. 5 per cent. susp. . 


... 0.5 j 0.5 0.5 

• 1 


0.5 


0.5 


0.5 


o.S 


0.5 


Normal saline 


. . . 1 .0 O.I O.I 


0. 1 


0. 1 


0. 1 


0. 1 


0. 1 







Technic for the Actual Test. — The patients' sera, the corpuscles 
and the antigen-complement-amboceptor-saline mixture are now 
ready for the test. The cholesterin antigen-complement-ambo- 
cep tor-saline mixture is contained in d, Fig. 39, and, inasmuch as 
there are usually positive cases even where a small number of 
tests are made, a second tube directly behind d should contain 
acetone-insoluble "a-c-a-s" mixture. If a gonococcus comple- 
ment fixation test is to be done then another tube placed in the 
rear of the block must contain gonococcus "a-c-a-s" mixture. 

1. For each Wassermann test to be done place a tube (120 X 13 
mm.) in the front row of a rack (Fig. 38). In next to the last hole 
of the series place a tube for a known positive serum and in the 
last hole a tube for a known negative serum. 

If a gonococcus test is to be done place a tube in the second 
row directly back of the one in the front row. 

2. Add to each tube 0.1 c.c. of patient's serum (inactivated 
thirty minutes at 54° to 55 ). Use for measuring, a pipette gradu- 
ated in hundredths to the very tip. Keep this pipette in a, Fig. 
39, and wash four times -between each serum by drawing it three- 
fourths full of saline and then blowing out into the sink. 

3. To each tube add 1 c.c. of cholesterin u a-c-a-s" mixture. 
If there is a gonococcus tube in the second row add, of course, 1 
c.c. of gonococcus u a-c-a-s" mixture to it. 

4. Incubate in water-bath thirty-five minutes at 37.5°C. 

5. Add 0.5 c.c. of 5 per cent, sheep corpuscles to each tube: 

6. Incubate in water-bath one hour at 37.5°C. 

7. Read results. 

8. For each positive Wassermann place a tube properly 
5 



66 



HOSPITAL LABORATORY METHODS 



Antigen-complement- amboceptor-saline Table 
Use i C.c. of this Mixture per Test 



No. of 


Antigen o.i c.c. 


Complement 0.05 


Amboceptor, 


Sterile normal saline, 


tests 


of proper dilution 


c.c. undiluted 


2 units 


q.s. ad 1 c.c. 


5 


0.5 c.c. 


0.25 c.c. 


.04—0.2 c.c. 
.06-0.3 c.c. 
0.08-0.4 c.c. 
0.1 -0.5 c.c. 
. 12-0.6 c.c. 
0.14-0.7 c.c. 


4.05 c.c. 
3.95 c.c. 
3.85 c.c. 
3-75 c.c. 
3.65 c.c. 
3-55 c.c. 


10 


1 .0 c.c. 


0.5 c.c. 


0.04-0.4 c.c. 
0.06-0.6 c.c. 
0.08-0.8 c.c. 
O.I -I .0 c.c. 

0.I2-I .2 C.C. 

0.14-1 .4 C.C. 


8.1 c.c. 
7.9 c.c. 
7.7 c.c. 
7-5 c.c. 
7-3 c.c. 
7.1 c.c. 


15 


1.5 c.c. 


0.75 c.c. 


0.04-0.6 c.c. 
0.06-0.9 c.c. 
0.08-1.2 c.c. 
0.1 -1.5 c.c. 
0.12-1 .8 c.c. 
. 14-2 . I c.c. 


12.15 c.c. 
11.85 c.c. 
11.55 c.c. 
11.25 c.c. 
10.95 c.c. 
10.65 c.c. 


20 


2.0 C.C. 


1.0 c.c. 


. 04-0 . 8 c.c. 

0.06-1 .2 C.C. 

0.08-1 .6 c.c. 

O.I -2.0 C.C. 
0. 12-2 .4 C.C. 

0. 14-2.8 c.c. 


16.2 c.c. 
15.8 c.c. 
15.4 c.c. 
15.0 c.c. 
14.6 c.c. 
14.2 c.c. 


25 


2.5 c.c. 


1.25 c.c. 


0.04-1 .0 c.c. 
0.06-1.5 c.c. 
0.08-2 .0 c.c. 
0.1 -2.5 c.c. 
0.12-3 c.c. 
0.14-3.5 c.c. 


20,25 c.c. 
19.75 c.c. 
19.25 c.c. 
18.75 c.c. 
18.25 c.c. 
17.75 c.c. 


30 


3.0 c.c. 


1.5 c.c. 


O.O4-I .2 C.C. 

0.06-1 .8 c.c. 
0.08-2 .4 c.c. 
0.1 -3.0 c.c. 
0. 12-3 .6 c.c. 

. I4-4. 2 C.C. 


24.3 c.c. 
23.7 c.c. 
23.1 c.c. 
22.5 c.c. 

21 .9 C.C. 
21 .3 C.C. 


35 


3-5 c.c. 


1.75 c.c. 


0.04-1 .4 C.C. 
0.06-2 .1 C.C. 
0.08-2.8 C.C. 
0.1 -3.5 c.c. 
0. 1.2-4.2 c.c. 
0.1*4-4.9 c.c. 


28.35 c.c. 
27.65 c.c. 
26.95 c.c. 
26.25 c.c. 
25.55 c.c. 
24.85 c.c. 


40 


4.0 c.c. 


2 .0 C.C. 


0.04-1 .6 c.c. 
0.06-2.4 c.c. 
0.08-3.2 c.c. 
0.1 -4.0 c.c. 
0. 12-4.8 c.c. 
0.14-5.6 c.c. 


32.4 c.c. 
31.6 c.c. 
30.8 c.c. 
30.0 c.c. 
29.2 c.c. 
28.4 c.c. 


45 


4.5 c.c. 


2 .25 C.C. 


0.04-1 .8 c.c. 
0.06-2 .7 c.c. 
0.08-3 -6 c.c. 
0.1 -4.5 c.c. 
0.12-5 -4 c.c. 
0.14-6.3 c.c. 


36.45 c.c. 
35.55 c.c. 
34.65 c.c. 
33-75 c.c. 
32,85 c.c. 
31.95 c.c. 



BLOOD 



6 7 



Antigen-complement- amboceptor-saline Table.- 
Use 1 C.c. of this Mixture per Test 



-Continued 



No. of 


Antigen 0.1 c.c. 


Complement 0.05 


Amboceptor, 


Sterile normal saline, 


tests 


of proper dilution 


c.c. undiluted 


2 units 


q.s. ad 1 c.c. 


SO 


5.0 c.c. 


2.5 c.c. 


0.04-2 .0 c.c. 
.06-3 .0 c.c. 
0.08-4.0 c.c. 
0.1 -50 c.c. 
0. 12-6.0 c.c. 
0. 14-7 .0 c.c. 


40.5 c.c. 
39.5 c.c. 
38.5 c.c. 
37-5 c.c. 
36.5 c.c. 
355 c.c. 


55 


5-5 c.c. 


2.75 c.c. 


.O4-2 . 2 C.C. 

0.06-3.3 c.c. 
0.08-4.4 c.c. 
0.1 -5.5 c.c. 
0.12-6.6 c.c. 
0.14-7.7 c.c. 


44-55 c.c. 
43-45 c.c. 
42.35 c.c. 
41.25 c.c. 
40.15 c.c. 
39.05 c.c. 


60 


6 .0 c.c. 


3.0 c.c. 


0.04-2 .4 c.c. 
. 06-3 . 6 c.c. 
0.08-4.8 c.c. 
0.1 -6.0 c.c. 
0.12-7.2 c.c. 
0.14-8.4 c.c. 


48.6 c.c. 
47.4 c.c. 
46.2 c.c. 
45.0 c.c. 
43.8 c.c. 
42.6 c.c. 


65 


6.5 c.c. 


3.25 c.c. 


0.04-2.6 c.c. 
0.06-3 -9 c.c. 
0.08-5.2 c.c. 
0.1 -6.5 c.c. 
0.12-7.8 c.c. 
0.14-9. 1 c.c. 


52.65 c.c. 
51.35 c.c. 
50.05 c.c. 
48.75 c.c. 
47.45 c.c. 
40.15 c.c. 


70 


7.0 c.c. 


3-5 c.c. 


0.04-2.8 c.c. 
0.06-4.2 c.c. 
0.08-5 -6 c.c. 
0.1 -7.0 c.c. 
0.12-8.4 c.c. 
0. 14-9.8 c.c. 


56.7 c.c. 
55.3 c.c. 
53.9 c.c. 
52.5 c.c. 
5 1. 1 c.c. 
49-7 c.c. 


75 


7-5 c.c. 


3-75 c.c. 


0.04 -3.0 c.c. 
0.06 -4.5 c.c. 
0.08 -6.0 c.c. 
0.1 -7.5 c.c. 

. 12 -9.0 C.C. 

0. 14-10. 5 c.c. 


60.75 c.c. 
59.25 c.c. 
57-75 c.c. 
56.25 c.c. 
54-75 c.c. 
53.25 c.c. 


80 


8.0 c.c. 


4.0 c.c. 


0.04 - 3.2 c.c. 
0.06 - 4.8 c.c. 
0.08 - 6.4 c.c. 
0.1 -8.0 c.c. 


64.8 c.c. 

63 .2 C.C. 

61 .6 c.c. 
60.0 c.c. 








0.12 — 9.6 C.C. 
0. 14 -II .2 C.C. 


58.4 c.c. 
56.8 c.c. 


85 


8.5 c.c. 


4.25 c.c. 


0.04 - 3.4 c.c. 
0.06 - 5.1 c.c. 
0.08 - 6.8 c.c. 
0.1 - 8.5 c.c. 

0. 12 -10.2 C.C. 

0. 14 -11 .9 c.c. 


68.85 c.c. 
67.15 c.c. 
65.45 c.c. 
63.75 c.c. 
62 .50 c.c. 
60.35 c.c. 


90 


9 .0 c.c. 


4-5 c.c. 


0.04 - 3.6 c.c. 
0.06 -5-4 c.c. 
0.08 -7.2 c.c. 
0.1 —9.0 c.c. 
0.12 -10.8 c.c. 
0.14 -12.6 c.c. 


72.9 c.c. 
71 .1 c.c. 
69.3 c.c. 
67.5 c.c. 
65.7 c.c. 
63.9 c.c. 



68 



HOSPITAL LABORATORY METHODS 



a 



numbered with blue pencil in the front row of a 
rack and directly behind it a second tube. The 
tubes in the front row are for acetone-insoluble an- 
tigen and those in the second row are for controls. 
Therefore with the tubes in the front row carry 
out the steps 1-7 inclusive using acetone-insoluble 
"a-c-a-s" mixture instead of the cholesterin. With 
the tubes in the second row carry out the steps 
1-7 inclusive but use u c-a-s" mixture (a mixture 
identical with the u a-c-a-s" mixture except that 
it contains no antigen) . No antigen is added to the 
control tubes in the second row. All second row 
tubes should show complete hemolysis. If they do 
not the test must be reported " anti-complementary." 
Even when only a single test is to be performed 
it is better to make up the "a-c-a-s" mixture as in- 
dicated. But in such a case the test with the ace- 
tone-insoluble antigen may be run in the third and 
the control in the second row, so that all three are 
run at the same time. In laboratories, however, 
where there are large numbers of tests to be per- 
formed it is a useless waste of complement to run 
controls on negative sera. 



Fig. 40. — Bac- 
terio logical 
s pecime n s . 
Sterile cotton 
swabs. Dry- 
heat sterilize 
and then auto- 
clave. Use one 
swab for making 
culture and the 
other for mak- 
ing smear on a 
slide, a, Wooden 
applicators ob- 
tained in boxes; 
b, small amount 
of cotton firmly 
wrapped by ro- 
tating a. 



BACTERIOLOGICAL SPECIMENS 

Introduction. — Material for cultures is most 
commonly sent to the laboratory on cotton swabs 
(Fig. 40). If the pus or other material is placed 
in a test-tube and presented for cultural purposes, 
the cultures may be made with a sterile platinum 
loop or if fluid with a sterile pipette. 

Whether the material to be examined bacterio- 
logically is sent on a swab, in a test-tube, or other- 
wise, it must be accompanied by a brown slip (Fig. 
41) filled out by the clinician. 



BACTERIOLOGICAL SPECIMENS 69 

REPORT FOR THE PATHOLOGIST 



Date 

Name 

Sex Age 

Service, Ward Bed . 

If not a hospital patient, give name and address of physician 



Duration and Nature of Disease. 



Place from which each piece of tissue for diagnosis is removed . 



Clinical Diagnosis 

Diagnosis of Pathologist. 



Signed, 



. Pathologist 



Instruction to House Officers 

All tissue must be immediately wrapped in sterile gauze, moistened with salt solution and 
then wrapped with oiled paper and the brown slip. 



Fig. 41. — Bacteriological Specimens. Brown slip. This slip of light brown 
paper 6^2 by 9M inches must be filled out by the clinician and sent to the laboratory 
with all surgical and bacteriological specimens. As soon as a diagnosis is made it is 
written on this slip which is at once returned to the clinician. 



70 HOSPITAL LABORATORY METHODS 

A number of systems for numbering and recording specimens 
are in use. Perhaps the simplest one is to indicate bacteriological 
specimens by B, surgical specimens by S, and autopsy specimens 
by A, and to place the last two numerals of the current year after 
these letters to show in what year the specimen was received. 
Following this system the first bacteriological specimen received 
in 191 6 bears the number B 16.1 and the 515th specimen bears 



B. 


Diagnosis: 


Material: 


Name: 


Service: 


Clinical Diagnosis: 


Smear (Stain) : 


DfltP i Rec eived 
Uate { Reported 


Agar: 






Litmus Milk: 









Fig. 42. — Bacteriological Specimens. Index card for bacteriological records. 
Cards 5 by 7 inches. All cards for indexing should be this size. 

the number B 16.515, etc. At the time the brown slip is removed 
from about the specimen the bacteriological number is written 
on the bottom of the slip. The consecutive numbers are kept on 
a sheet and as each specimen is given a number, this one is crossed 
off on that sheet. The number is also placed on a card (Fig. 42) 
for the laboratory record. 

As a routine two swabs are sent to the laboratory. With one 



BACTERIOLOGICAL SPECIMENS 



71 



of these a smear is made on a slide and stained by Gram's method. 
With the other a culture is made first on an agar slant by gently 
rubbing it over the agar surface and then in a tube of litmus 
milk. - Other media are used if indicated. 




Fig. 43. — Bacteriological Specimens. Rack for bacteriological cultures 
Wooden test-tube rack for sixteen culture tubes. In racking up a number of 
separate cultures, place agar-slant cultures in front row and the litmus-milk cul- 
tures in back row. 



p. |K a r-v p\ p 



X 



The bacteriological number (Example: B 16.515 or just 515) 
is written on each inoculated tube with a pencil. The cultures are 
placed in a rack (Fig. 43) with the agar cultures in the front row. 
The rack is placed in the incu- 
bator at 37 . The following 
morning a smear is made from 
each of the agar slants and each 
milk culture and stained by 
Gram's method (p. 79). In ex- 
amining colonies from a plate or 
a large number of tube or flask 

cultures it is very convenient Fig. 44.— Bacteriological ^ Speci- 

b , 1£ENS. Slide with eight preparations, a, 

to make about eight prepara- Cross made with a wax pencil. The prep- 

4-:~~„ ~~ ~ j-j„ /-[?• \ 1 aration next to this cross is number one. 

tions on a slide (Fig. 44), mark- ^ Eight small drops of water placed on 

ing the exact position of each the slide with pipette, Fig. 56. Add the 

. , , , . . material from the cultures with platinum 

With a blue pencil at the margin loop and spread each out as indicated. 

of the slide. Place preparation ^ Iark th ? exact Potion of each prepara- 

r r tion on the margin c with a wax pencil. 

No. i at the left end of the slide. 

The cards (Fig. 42) are kept during the current year in a 



72 HOSPITAL LABORATORY METHODS 

card index case in the order of the numbers on them. The cards 
for previous years may be tied together and filed away. There 
must be a card for each number. As soon as ascertained, the 
diagnosis is written on the brown slip which is signed and returned 
to the clinician. 

Sterile swabs (Fig. 40) are best prepared by wrapping one end 
of the wooden throat applicator firmly with cotton, placing two 
of these in a test-tube and plugging the upper end of the tube 
with cotton. The tubes are dry-heat sterilized and then auto- 
claved. The lower ends of applicators should reach the bottom of 
the tubes and the upper ends should project one inch or more. One 
of the swabs is used for making direct smears on slides and the 
second is used for making cultures. 

For routine diagnosis, the culture media and the stains should 
be made as simple as possible. The details of the preparation 
and the application of the media and stains used should be thor- 
oughly understood. 

Media. — The number of bacteria present may be so small that 
they cannot be found in smears of the material; it may be im- 
possible to identify organisms from smears alone; or it may be 
desirable to test the biologic characters of the organism. In 
such cases the organisms must be grown on media in cultures and 
often in pure cultures. It is also necessary to grow the organisms 
on artificial media for the preparation of vaccines, antigens, etc. 

Plating. — For the isolation of bacteria in pure cultures, the 
individual bacteria are mechanically separated from each other 
to a distance of at least 1 cm. This may be done by mechanically 
rubbing the bacteria-containing substance over the surface of a 
solid medium, or more often by mixing it with melted agar (agar 
melted at ioo° and cooled to 45 ) that becomes solid on cooling. 
The melted agar after inoculation is usually poured into a Petri 
dish so as to get a flat surface. The single separate bacterial 
cells multiply in the medium sufficiently to give rise to a macro- 
scopic mass, a colony. Such a colony of course consists of a single 
kind of organism as it comes from a single bacterial cell. 

Tube Cultures. — For obtaining cultures that are not necessarily 



BACTERIOLOGICAL SPECIMENS 



73 



pure and for making subcultures from colonies and other pure 
cultures, media in tubes are usually employed. 

Agar. — Dissolve 5 gm. Liebig's meat extract, 5 gm. salt and 10 
gm. peptone in 1000 c.c. of water at 55°C. Titrate 0.8 per cent, 
acid. 











Fig. 45. — Bacteriological Speci- Fig. 46. — Bacteriological Speci- 
mens. Funnel for filtering media, a, mens. Funnel for tubing media, a, 
20-cm. funnel; b, large cork notched with 15-cm. funnel; b, soft rubber tubing; 
knife; c, thin layer of cotton; d, iron ring c, pinch cock; d, glass tip. 
supporting funnel. 

The titration is -made on 5 c.c, removed with a graduated 
pipette and placed in a small porcelain dish. Add 20 c.c. of dis- 
tilled water and boil for one minute. While hot add 2 drops of 
0.5 per cent, alcoholic solution of phenolphthalein and run in 



74 



HOSPITAL LABORATORY METHODS 



twentieth-normal sodium hydroxide until the first distinct pink 
appears. The number of cubic centimeters of sodium hydrate 
used multiplied by 10 gives the number of cubic centimeters of 
normal alkali required to neutralize i liter. Suppose 1.5 c.c. of 
the twentieth-normal hydrate are used for the 5 c.c. then 1.5 X 10 
= 15 c.c. of N. alkali required to neutralize 1 liter. With this 
titration, to make the liter 0.8 per cent, acid (0.8 per cent, of 
1000 = 8), add 7 c.c. (15 c.c. — 8 c.c. = 7 c.c.) of normal hy- 
droxide. 




Fig. 47. — Bacteriological Specimens. Slants of solid media. The upper 
portion of the slant a should reach to within 1 inch of the lowermost part of the 
cotton plug b. 

After adjusting the reaction add 30 gm. of pulverized agar 
or 20 gm. unpowdered agar, boil ten minutes, make up to 1000 
c.c, cool the medium to 45°, and thoroughly stir in the white of 
one egg. Raise slowly to the boiling point and boil gently for 
five minutes without stirring or agitating the mixture. The egg 
coagulates in large masses and the clear liquid readily runs 
through muslin placed in a large funnel provided with a cork at 
its opening. A thin layer of cotton is placed over the muslin. 
The hot agar is poured on only so fast as it filters through 
(Fig. 45). 

For slants, fill the tubes from the funnel used for tubing (Fig. 
46) to such a height that the upper edge of the slant lacks 1 inch of 



BACTERIOLOGICAL SPECIMENS 75 

reaching the cotton plug, while the bottom of the tube is not 
completely covered with medium (Fig. 47). 

To make glycerine agar from the plain agar, add 2 per cent, of 
twice distilled glycerine to the desired quantity of melted agar in 
a flask, mix and tube. For glucose agar add 2 per cent, glucose. 

Arnoldize all sugar media and gelatin and autoclave all other 
simple media at 1 io°C. for twenty minutes. All glassware cleaned 
and plugged with cotton or placed in containers must be heated 
in the dry-heat oven at 170 to 2oo°C. for fifteen minutes. 

To prepare blood agar from the plain sterile slants, add about 
20 per cent, of sterile defibrinated rabbit or human blood from a 
pipette to the agar tubes melted and cooled to 45°C. Thoroughly 
mix by rolling. Slant. The sterile non-defibrinated blood from 
the arm vein of a patient may be added directly from the syringe. 
To prepare hydrocele or ascitic agar, add these fluids in the same 
way that the blood was added. All slants should be placed in 
the incubator for twenty-four hours to test their sterility and 
they should be kept permanently in the slanted position until 
used. The preparation of these media differs from the preparation 
of glycerine and sugar media in that the material added must be 
kept sterile. To do this a sterile bulb pipette (Fig. 36) is very 
useful but sterile 10 c.c. pipettes (h, Fig. 33) may be used. 

Bouillon. — Dissolve 5 gm. Liebig's meat extract, 5 gm. salt 
and 10 gm. peptone in 1000 c.c. water. Boil fifteen minutes, make 
up to one liter, titrate to 0.8 per cent, acid, autoclave at no° for 
twenty minutes, and filter through filter paper. Place in flasks 
or tubes and autoclave at no°C. for twenty minutes. 

For the cultivation of some bacteria, it is desirable to obtain 
the meat extractives directly from the meat. In cases where the 
laboratory facilities are good it is advisable to use chopped beef 
as a routine. To use the beef, "1000 c.c. of beef infusion' 7 is 
substituted in the directions for "5 gm. Liebig's meat extract " 
and the liter of water omitted. 

The "beef infusion" is prepared by adding 1000 c.c. of water to 
500 gm. chopped, spice-free lean beef (beef ground in a sausage 
grinder), placing in the ice-box for twenty-four hours and then 



7 6 



HOSPITAL LABORATORY METHODS 



filtering through muslin. The filtrate is the "beef infusion." 
Another way of preparing the " beef infusion" is to heat at 55°C. 
for one hour instead of allowing to stand for twenty-four hours 
in the ice-box. At times heat may remove desirable substances 
from the infusion. This may be avoided by adding the peptone 
and salt to the infusion, correcting the reaction and then passing 
through a Berkefeld filter (Fig. 48). 




Fig. 48. — Bacteriological Specimens. Berkefeld filter, a, Connects with pipe 
from compressed air apparatus; b, ordinary steam gage; c, heavy metal or rubber 
pipe. If c is rubber tubing then a small connection d must be screwed into the upper 
end of the metal case e. The bougie is attached to the milled end/. The filters are 
obtained from Aug. Giese and Son, 4 Cedar St., New York City, g is a sterile flask 
for reception of the filtrate. An excellent copper case (e) for pressure filtration may 
be obtained from Isaac Young and Co., Coppersmiths, East Boston. Suction may 
be used for filtration. 



Litmus Milk. — To 1 liter of " fat-free " sweet milk, add suffi- 
cient 1 per cent, azolitmin to give the desired blue. 

Gelatin. — To 1000 c.c. water add 5 gm. Liebig's beef • extract, 
5 gm. salt and 10 gm. peptone and dissolve at 55°C. Make the 
gelatin neutral to phenolphthalein (see titration of agar) ; cool to 
3o°C. and thoroughly stir in the white of one egg; boil for five 



BACTERIOLOGICAL SPECIMENS 



77 



minutes without stirring; filter through muslin and a thin layer of 
cotton. Tube. 

Dunham's Solution. — To 1000 c.c water add 10 gm. peptone 
and 5 gm. salt. Dissolve with heat and filter through paper. 

Lactose Bile. — Autoclave one liter fresh ox-bile, filter, add 
two per cent, lactose, place in tubes and flasks and fractional 
sterilize. 

Serum Water. — To 250 c.c. calf or pig serum, add 750 c.c. 
water. Add 1 per cent, azolitmin to pale blue color and 1 per 
cent, dextrose, mannose, lactose, saccharose, or inulin. Of these 
sugars the first two are monosaccharids (aldehyde-alcohols with 
six carbons), lactose and saccharose are disaccharids (former d 
glucose + d galactose and w T hile the latter is made up of two d 
glucose molecules). Inulin is a polysaccharid. The approxi- 
mate way (there are variations within the groups) in which organ- 
isms attack these sugars is shown in the attached table. 



Inulin 



Saccharose 


Lactose 


Mannose 


Dextros 


Few strains 


Gas 


Gas 


Gas 





Acid 


Gas 


Gas 








Gas 


Gas 








Gas 


Gas 








Gas 


Gas 








Acid 


Acid 








Acid 


Acid 











Acid 














Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 


Acid 



B. Coii o 

B. Mucosus capsulatus o 

B. enteritidis o 

B. Paratyphosus B o 

B. Paratyphosus A ; o 

B. Typhosus o 

B. Dysenteriae (Flexner) o 

B. Dysenteriae (Shiga) o 

Micrococcus meningitidis o 

Strep, pyogenes o 

Strep, of subacute endocarditis. . o 

Strep, mucosus cap Acid 

Strep, pneumoniae Acid 

Staph, pyogenes aureus Acid 



Loeffler's Blood Serum.- — To 250 c.c. 1 per cent, glucose 
bouillon, add 750 c.c. calf or pig serum. Tube, slant, place in 
dry-heat oven or in an inspissator and very slowly raise the tem- 
perature to 85°C. and maintain that temperature for two hours. 
Autoclave. 

Drigalski Medium. — Make 1 liter of agar in the usual way 



7 8 



HOSPITAL LABORATORY METHODS 



except 10 gm. of nutrose are added and it is made neutral to phenol- 
phthalein. To the melted agar add i per cent, of lactose, i per 
cent, of a o.i per cent, solution of crystal violet and enough i per 
cent, solution of azolitmin to give the desired color. Autoclave. 

Petroff Medium for Isolation of Tubercle Bacilli. — To the 
material (sputum) in a centrifuge tube of suitable size (50 ex.), 
add an equal amount of normal sodium hydroxide, stir thoroughly 
and place in the incubator at 37°C. for thirty minutes. Add 
normal hydrochloric acid to neutralize exactly the normal alkali 
used. Centrifuge and make smears or cultures from the sediment. 

To prepare the medium, place eggs in alcohol for one-half hour, 
break into a sterile beaker and thoroughly beat. The beaten egg 




Fig. 49. — Bacteriological Specimens. Block for stains. Bottles hold 30 c.c. 
and are obtained from Bausch and Lomb Optical Co. The block consists of a lower 
%-inch board 25 by 3.5 inches k on which there is tacked a board, Z, that has in it 
ten 1.5-inch holes for the bottles, a, Gram's staining solution; b, Gram's iodine; c y 
95 per cent, alcohol; d, pyronin; e, Ziehl-Neelsen;/, Czaplewsky; g, Loeffler's methy- 
lene blue; h, distilled water; i, polychrome stain for protozoa;,;, polychrome blood 
stain. 

is then measured in a sterile graduate. To 2 parts of the sterile 
egg, add 1 part of sterile glycerinated beef juice. 

To prepare the sterile beef-juice, add 500 c.c. ^15 per cent, 
glycerine to 500 gm. chopped beef and allow to stand over night. 
Filter through gauze, add 15 c.c. of 1 per cent, alcoholic solution 
of gentian violet and pass through a Berkefeld filter (N) into a 
sterile flask. After mixing the egg and beef-juice in the sterile 
flask, pour into a sterile funnel tubing apparatus and tube into 
sterile test-tubes. Place the sterile egg-beef-juice mixture in 



BACTERIOLOGICAL SPECIMENS 79 

tubes, slowly raise the temperature to 85°C. and hold it at that 
temperature for two hours. Autoclave.* 

Stains. — The number of stains used should be reduced to a 
minimum and the worker should familiarize himself with every 
detail.of these. A ten-hole block (Fig. 49) for ten 30 c.c. staining 
bottles with snout-leaks is very convenient: (1) Gram's Staining 
Solution, (2) Gram's Iodine, (3) Pyronin, (4) Loeffler's Methylene 
Blue, (5) Ziehl-Neelsen, (6) Czaplewsky, (7) 95 per cent. Alcohol, 
(8) Blood Stain, (9) Polychrome Stain and (10) Distilled Water. 

Write the labels and when dry apply thin celloidin with a 
small brush. As soon as this is dry paint over with balsam damar 
in xylol. The balsam damar in xylol is obtained from Bausch 
and Lomb. 

Unless otherwise directed all bacterial preparations are fixed 
in the flame. Proper fixation must be acquired by experience. 
To fix a smear on a slide, hold the slide for ten seconds with the 
preparation side up 3 inches above a Bunsen burner flame 4 inches 
high. If the preparation is on a cover, hold the cover-glass an 
inch or two farther above the top of the flame for the same length 
of time. 

Loeffler's Methylene Blue. — To 1485 c.c. of distilled water, 
add 500 c.c. of a saturated absolute alcoholic solution of methylene 
blue (to saturate place a bottle containing 2000 c.c. absolute 
alcohol and 30 gm. methylene blue in the paraffin oven for several 
hours and shake repeatedly) and 15 c.c. of 1 per cent, aqueous 
solution of potassium hydroxide. It stains in a few seconds 
and does not readily overstain. This stain brings out as much 
detail in the morphology of Klebs-Loeffler bacillus as any of the 
special stains. 

Gram's Stain. — Two stock solutions are required: Solution A 
is made up by adding an excess of methyl violet to 330 c.c. absolute 
alcohol and 90 c.c. aniline oil in a 500-c.c. glass-stoppered bottle. 
Solution B is made by adding an excess of methyl violet to 400 
c.c. of distilled water in a 500-c.c. glass-stoppered bottle. 

To make the staining solution (Gram's staining solution), add 
2 c.c. of A to 18 c.c. of B in a 30-c.c. staining bottle provided with 



8o 



HOSPITAL LABORATORY METHODS 



a snout-leak. The Gram's staining solution keeps two weeks. 
For this reason it is necessary to make the solution up fresh from 
the two stock solutions once in two weeks. Solutions A and B are 
permanent. 

The fixed preparation is covered with the Gram's staining 
solution for three minutes. Wash off with water and apply Gram's 
iodine (iodine i gm., potassium iodide 2 gm. and water 300 c.c.) 
for thirty seconds. Wash the iodine off with water and then run 
95 per cent, alcohol over the preparation for thirty seconds. 
Wash with water and stain with pyronin (pyronin 2 gm., 10 per 











jf^ 




f\ 




>\ 




$ 


aU_. 


^ 






x jj 


- c 




V 





Fig. 50. — Bacteriological Specimens. Support for tubercle bacillus stains 
(Barta). a, Rubber tubing provided with a cock and leading to an elevated water 
bottle; b, rubber tubing leading into sink; c, box of thin galvanized iron nailed 
against the wall; d, wire on which the slide is placed; the slide is heated with a 
Bunsen flame by occasionally waving the flame back and forth beneath it. 



cent, formalin 10 c.c. and water 90 c.c.) for one minute. Wash, 
air dry and examine with the oil. Each individual must test the 
time for each step in the stain by using slides that have on them 
both Gram negative and Gram positive bacteria. 

Ziehl-Neelsen Stain for Tubercle Bacilli. — To 450 c.c. of 5 
per cent, carbolic acid in a glass-stoppered bottle, add 50 c.c. of a 
saturated absolute alcohol solution of fuchsin (neutral). To 
saturate the absolute alcohol with fuchsin place in tightly stop- 
pered bottle in paraffin oven and shake. A stock bottle of the 
saturated fuchsin solution should be kept on hand. 



BACTERIOLOGICAL SPECIMENS 



8l 



Czaplewsky's solution for decolorizing the tubercle bacilli 
stains is made by adding 5 c.c. of concentrated hydrochloric acid, 
5 gm. of sodium chloride and 200 c.c. of water to 1000 c.c. of 95 
per cent, alcohol. 

The smear properly fixed on a slide is placed on a staining 
support (Fig. 50), the Ziehl-Neelsen solution placed on the prepara- 
tion and heated by occasionally. waving a Bunsen flame beneath 




■■/ 



Fig. 51. — Bacteriological Specimens. Guinea-pig holder, a, Board 18 
inches long by 8 inches wide supported by two ends, /, 8 inches wide by 6 inches 
high; b, hole through which leather thong c passes. The slip-knots are passed over 
the hind legs after the wooden strip e has been fastened so as to hold the head in 
the wooden block d. 



it so as to cause steaming for two to five minutes. Apply fresh 
stain as often as 'necessary. The slide may also be heated by 
placing it across the rings of a boiling water-bath. Wash off the 
stain by flooding the preparation with water. The Czaplewsky 
solution is now run over the preparation by dropping from the 
bottle until the thinner parts of the smear are only a very faint 
pink. Wash with water and stain with Loeffler's methylene blue 
for one-half minute. Wash, air dry and examine with oil. The 

6 



82 



HOSPITAL LABORATORY METHODS 



tubercle bacilli are red while all other parts of the preparation are 
blue unless there are heavy masses in the smear which may retain 
the red. 

Capsule Stain. — Stain with Mcjunkin's polychrome stain for 
protozoa after fixation in absolute alcohol-ether. 

Flagellum Stain (Lowit). — Fix in 10 per cent, formalin for one 
hour. Great care must be taken. to preserve the flagella in making 
the preparation. Place the freshly prepared mordant (10 c.c. of 
20 per cent, tannic acid, 5 c.c. saturated aqueous solution of copper 
sulphate and 1 c.c. of saturated alcoholic solution of fuchsin 




Fig. 52. — Bacteriological Specimens. Autopsy board, a, %-inch board 
of soft wood large enough for rabbit; b, small wire nails. 



(neutral) ; filter) on the preparation for three minutes ; wash and 
stain five minutes with the Gram's staining solution steaming 
gently. Wash, air dry and mount in balsam. 

Spore Stain. — To establish the presence of spores with cer- 
tainty it is necessary to make a differential stain. (1) Young 
spores: Fix in flame. (2) Five per cent, chromic acid for ten min- 
utes. Wash. (3) Carbol-fuchsin, steaming gently for ten min- 
utes. Wash. Examine in water. The spores must be red. (4) 
Differentiate in 95 per cent, alcohol until only the spores remain 
red. Wash and examine in water. (5) Stain with Loeffler's 
methylene blue. Dry and mount in balsam. 

Special Specimens. — Some bacteriological specimens sent to 
the laboratory require a special technic in order to determine the 



BACTERIOLOGICAL SPECIMENS 



83 




organisms present. Specimens of urine from catheterized ureters 
are frequently requested to be used for guinea- 
pig inoculation. In cases where the gas bacillus 
or tetanus is suspected, a special procedure is re- 
quired. The request for such special examinations 
must always be written on the brown slip by the 
clinician. 

Cerebrospinal Fluid. — Cloudiness, if no 
blood is present, suggests meningitis. If fresh make 
a preparation in the blood-counting chamber with 
the undiluted fluid. The number of cells in 1 c.c. 
should not exceed twenty. 

Centrifuge in a 15-c.c. sterile centrifuge tube, 
remove the sediment from the bottom with a sterile 
capillary pipette and plant the sediment on blood 
serum, ascitic agar and blood agar; incubate and 
examine the cultures daily. Also make a smear 
from the sediment and stain with polychrome stain 
for blood for a differential count of the cells. 

Estimate the globulin in the clear supernatant 
fluid by adding 0.5 c.c. of 10 per cent, butyric acid 
to 0.2 c.c. of the cerebro-spinal fluid in a small test- 
tube. Heat and add 0.1 c.c. of normal sodium hy- 
drate. A precipitate indicates globulin (Noguchi). 

If a Wassermann is done use 1 c.c. of the fluid, 
instead of 0.1 c.c. which is the usual amount of the 
patient's blood-serum used for this test. 

Fluid for Tubercle Bacilli. — Centrifuge in tube. Place 
a sterile centrifuge tube. Decant supernatant fluid. 



Fig. 53.— 
b a c t e riologi- 
cal Specimens. 
Fermentation 



test-tube a up- 
side down in the 
bouillon in the 
1 5 5 by 16-mm. 
test-tube b before 
sterilization. 
The air is driven 
out of a during 

c.c. of the sediment remaining in the t h e sterilization. 



Stain sediment by the Ziehl-Neelsen method. If 
there are no tubercle bacilli and few streptococci 
or none at all, a guinea-pig may be injected (Fig. 
47) on the request of the clinician. 

Inject 

bottom of the centrifuge tube subcutaneously in 
the right groin of a 250-gm. guinea-pig and % cx - intraperi 



84 



HOSPITAL LABORATORY METHODS 




Fig. 54. — Bacteriologi- 
cal Specimens. Anaerobic 
culture cylinder, a, 100-c.c. 
graduate; b, piece of cork 
to support culture tube; c, 
culture tube; d, upper end of 
graduate; e, tightly fitting 
stopper; /, paper wrapped 
about the upper end of the 
cylinder into which the paraffin 
is poured. 

of which there is 1 gm. 



toneally. Autopsy (Fig. 52) at the 
end of six weeks. Examine the tissue 
of the right groin, the spleen, liver, 
peritoneal surfaces and the lungs for 
tubercles by making smears on slides 
and staining for tubercle bacilli and 
by making colloidin sections. 

PUS OR CURETTINGS FOR B. AERO- 

genes Capsulatus. — Smears show a 
Gram positive bacillus about the size 
of the anthrax. Capsules are often 
demonstrable; the bacilli rarely form 
long chains. If the material is plenti- 
ful inject 0.5 c.c. into the ear vein of 
a rabbit, kill the animal after five 
minutes and place in the incubator for 
twelve hours. If the gas bacillus is 
present, the organs and cavities will 
contain large amounts of gas. 

If only a swab has been sent for 
diagnosis, inoculate two tubes of litmus 
milk and two of glucose agar. Place 
the litmus milk cultures under anaerobic 
conditions. After twelve hours inject 
0.5 c.c. of the litmus milk into the ear 
vein of the rabbit; kill and incubate as 
above. 

Material for Tetanus. — Examine 
smears for the Gram positive pin-shaped 
bacilli. Inoculate a dextrose bouillon 
fermentation test-tube (Fig. 53) and 
make a dextrose agar stab. Make the 
cultures anaerobic by placing the tube 
in a 100-c.c. graduate (Fig. 54) or other 
cylinder of heavy glass in the bottom 
(estimated) of pyrogallic acid. Add 10 



BACTERIOLOGICAL SPECIMENS 



85 



c.c. of 10 per cent, sodium or potassium hydroxide and stratify 
a layer of paraffin above and around the cork by holding a cylinder 
of paper snugly about the cork and upper end of cylinder and run- 
ning the melted paraffin into it. Examine daily. If plates or a 
number of tube cultures are desired, use the Novy anaerobic jars. 

Milk. — To sterile, wide-mouth bottles containing 99.5 c.c. of 
sterile water, 0.5 c.c. of the milk collected in sterile tubes is added. 

The milk is placed in the test-tubes by pieces of sterile glass 
tubing (3 feet long) kept sterile by wrapping in sterile muslin. 
These long pieces of tubing are used for stirring the milk in the can 
before the sample is placed in the sterile test-tubes in which the 
samples are taken to the laboratory. 

One cubic centimeter of this 1-200 dilution is added to one 
tube of melted agar cooled to 45°C. After mixing pour into a Petri 




Fig. 55. — Bacteriological Specimens. Petri dish ruled for counting colonies, 
a, Bottom of inverted Petri dish ruled off with a wax pencil into 9 parts. 

dish and incubate for forty-eight hours; 0.2 c.c. of the diluted 
milk is used for making a second plate. Count the colonies after 
forty-eight hours of incubation. Report the number of colonies 
per cubic centimeter of milk. Examine small translucent colo- 
nies for streptococci. In counting the colonies, rule the bottom 
of the plate into 9 parts with a wax pencil (Fig. 55). 

Nose and Throat Cultures for Diphtheria Bacilli. — With 
a swab (Fig. 40) rub the suspicious portion of the mucous membrane 
and with the swab inoculate a tube of Loeffler's blood serum. In- 
cubate over night and stain the eighteen-hour growth withLoeffler's 
methylene blue. Where a large number of cultures are examined 
daily, one may make eight preparations on a slide. A pipette 
with a double curve (Fig. 56) is very convenient for placing the 



86 



HOSPITAL LABORATORY METHODS 



8 small drops 




Fig. 56. — Bacte- 
riological Speci- 
mens. Pipette for 
placing minute 
drops of water 
on a slide. Made 
by heating a piece 
of glass tubing with 
a 3-mm. lumen in 
a flame, drawing 
the heated portion 
out and curving it 
as indicated. One 
or 2 c.c. of water is 
placed in the pip- 
ette from the open 
end a. b is touched 
to the slide at the 
points at which the 
small drops are de- 
sired. - 



of water on the slide to which the bacteria from 
the surface of the tube are added by means of 
the usual platinum loop. 

When the bacilli persist in the nose or throat 
for more than six weeks a test of the virulence 
of the organisms may be desirable. To do this 
make a swab from a culture that shows numer- 
ous bacilli of typical morphology and thoroughly 
wash the swab off in the water of condensation 
at the bottom of the tube of blood serum. Tubes 
that are the same length, but twice the diameter 
of an ordinary test-tube, are the best for this 
purpose. Repeat the process with the same swab 
on tubes numbers 2, 3, 4, 5, and 6, passing from 
one tube to another with the same swab. Flood 
the surface of the media of each tube after mak- 
ing the dilution and then incubate in the up- 
right position over night. Examine for diphtheria 
bacilli a number of translucent colonies from the 
surface of the slants that show well-isolated 
colonies and inoculate five separate tubes of 
blood serum from that many positive colonies. 
The diphtheria colonies are determined by mak- 
ing smears on slides (eight on a slide) and stain- 
ing with Loeffler's methylene blue. 

After twenty-four hours inoculate each of 
these tubes containing pure cultures into a 
350-c.c. Erlenmeyer flask containing 50 c.c. of 
bouillon. After forty-eight hours' incubation, 
inoculate a small guinea-pig subcutaneously with 
0.5 per cent, of its weight of the forty-eight-hour 
flask culture. 

If the guinea-pig dies within five days go back 
to the original five tubes containing the five pure 
cultures and inoculate another flask of bouillon 
with these. Incubate forty-eight hours and in- 



BACTERIOLOGICAL SPECIMENS 



87 




ject a second guinea-pig with 0.5 per cent, of its weight but along 
with the toxin, inject 0.5 cc. of antitoxin. If the pig lives five 
days the bacilli are diphtheria bacilli. 

Bacterial Vaccines. — To prepare a vaccine, open up the 
lesion freshly and make a swab (Fig. 40) with which an agar culture 
is made. If the culture is contaminated with organisms that are 
not desired in the vaccine, make a swab from the surface of the 
mixed agar culture and with this swab make 
dilutions on the surface of five agar slants as 
was done on ten blood-serum tubes with the 
diphtheria bacilli. The tubes are placed in 
the incubator. The following day colonies are 
examined and agar subcultures made from the 
desired ones. After incubation the tubes are 
filled half -full with saline and the cultures rubbed 
off with a platinum needle. Place the suspen- 
sion in a single sterile tube and heat for one 
hour at 6o°C. Make a culture to test the 
sterility of the suspension. 

To determine the number of bacteria in the 
suspension, place about 1 cc. of the sterilized 
suspension in a watch crystal and fill the blood 
pipette used for counting the red blood cor- 
puscles to the 0.5 mark with the suspension and 
then fill the pipette to 10.1 mark with Hay em's 
solution to which sufficient acid fuchsin (dry) 
has been added to give the solution a distinct red color. After 
the preparation on the slide has stood for one-half hour, count 
the bacteria the same as the red blood corpuscles are counted. 

Suppose there are 1 million in 1 c.mm., then 1 cc. will contain 
1000 million and 0.5 cc. will contain 500 million, which is an 
average dose of staphylococcus for example. Dilute the suspension 
with normal saline so that 1 cc. contains 1000 million bacteria 
per cubic centimeter. Add 0.5 per cent, phenol and place in 
10 cc. square sterile bottles and cover with a rubber cap (Fig. 57). 

Before using, this rubber cap is wiped off with lysol and the 



Fig. 57.— Bac- 
teriological Spec- 
imens. Vaccine 
bottle. a io-c.c. 
bottle; b f rubber 
cap. The bottle 
and the detached 
cap are autoclaved. 
Wipe off the cap 
with cotton mois- 
tened with lysol and 
insert the sterile 
needle through it to 
draw'up the desired 
amount of vaccine. 
Along i-c.c. vaccine 
syringe is used. 



88 



HOSPITAL LABORATORY METHODS 



needle of a long i-c.c. syringe (graduated in tenths) inserted 
through it. 

A case of furunculosis may for example be given ioo million 
one day , and after three days 200 million and up to 2000 million. 
Such vaccines are of ten given twice per week. 



SURGICAL SPECIMENS 

Introduction. — To properly care for hospital laboratory work, 
it is necessary to have a room (clinical laboratory) in which urine, 

blood, gastric contents, feces, 
sputum and certain body fluids 
are examined. These exami- 
nations are usually carried 
out more or less under the 
direct charge of the clinician. 
It is not usually required to 
keep a laboratory record of 
these examinations as they are 
p filed with the clinical histories 
by the examiner.' 

In small hospitals it may 
be necessary to care for the 

Fig. 58.— Surgical Specimens. K flr t Pri - n i n(n Val cnprimpn* snd 
Surgical tag numbers, a, Small block of bacteriological specimens ana 

wood into which a long wire nail b has the Surgical and autopsy tissue 
been driven. The tags are merchandise . . . 

tags (white) measuring 2 by i}i inches, m the Same room. In this 

The ^fixation is indicated in the lower right- case ^ ap p aratlis f or the 
hand corner. The numbers are consecutive. rr 

bacteriology and for handling 
the tissue should be separately placed in different parts of the 
room, and arranged so that large or small numbers of specimens 
may be handled in an orderly way. 

In large hospital laboratories, aside from the clinical laboratory, 
there is at least one room for the bacteriological work, one for the 
tissue work and one for autopsies. The bacteriological specimens 
are examined and the examinations recorded as indicated under 
that heading. 




SURGICAL SPECIMENS 89 

A diagnosis on tissue removed during the life of a patient is 
often desired as soon as it can be made, while the ten days required 
for the slow paraffin method is no disadvantage with tissue re- 
moved at autopsy. For this reason tissue received at the labora- 
tory should be divided into surgical specimens that are removed 
during life and that receive a surgical number and autopsy speci- 
mens that are removed after death and receive an autopsy number. 
The technic of handling surgical and autopsy tissue is often the 
same. 

Surgical specimens are assigned a number as soon as they reach 
the laboratory and are disposed of in the ways described below. 
Tags bearing consecutive numbers are kept on a file for this assign- 
ment of numbers (Fig. 58). The numbering is the same as that 
used for the bacteriological specimens; for example, S 16. 461 is 
the 461st surgical specimen received in 191 6. 

Wood Stain. — Desk- tops, table- tops, blocks for staining 
bottles, and other woodwork should be stainted black. To do 
this apply with a brush copper sulphate 125 gm., potassium 
chlorate 125 gm., and water 1000 c.c. and allow to dry. Cover 
with a second coat, allow to dry and apply aniline hydrochloride 
150 gm. and water 1000 c.c. and allow this to dry. Wash with 
soap and water and after drying, apply linseed oil which should 
be thoroughly rubbed in. Desks should be cleaned off with a 
cloth moistened with equal parts linseed oil and chloroform once 
per week. 

Frozen Sections for Immediate Diagnosis. — The sections 
that may be cut from the fresh tissue and the stains that may be 
made on these sections give results inferior to those obtained by the 
colloidin or paraffin method. However, the surgeon may require 
a diagnosis while the patient is under the anesthetic. In this case 
tissue is removed and immediately brought to the laboratory by 
an attendant. 

A piece of tissue not larger than 2 or thicker than 1 cm. is cut 
from the specimen delivered at the laboratory and placed on the 
disk of the freezing microtome (Fig. 59) . The liquid carbon dioxide 
is allowed to flow into the copper tube (e) by turning the valve (d) 



9° 



HOSPITAL LABORATORY METHODS 



on the lower end of the cylinder with a long-handled wrench. The 
carbon dioxide escapes against the disk on releasing the valve (/) 
on the disk end of the copper tube. Let the gas escape until the 
freezing zone shown by the whitening of the tissue just reaches 
the top of the block of tissue. 




Fig. 59. — Surgical Specimens. Freezing microtome, a, Chain or leather 
strap; b, CO2 tank; c, i-inch board with a 4.5-inch hole; c is 8 inches above,; into 
which the microtome is screwed; e, copper pipe with a cock, /, on the microtome 
end (Bausch & Lomb Opt. Co.); d, piece of metal pipe attached to the usual short 
handle wrench that comes with tube; g, glassway for knife; k, micrometer screw. 
The cylinder should be placed in the upright position otherwise gas and not liquid 
flows into e as soon as a horizontal cylinder is half empty. 

With the knife (Fig. 60) even off the upper surface to the 
place from which the sections are to be cut. If the tissue is not 
sufficiently thawed, blow gently on the block and just as soon 
as thawing begins on the surface rapidly cut a number of 
sections by turning the milled wheel (k) as the knife is 
rapidly slid back and forth. The sections are removed from 
the knife blade by dipping the knife into water in a finger bowl. 



SURGICAL SPECIMENS 9 1 

A slide is placed into this dish and one of the good sections 
floated onto it and the excess of water removed with a cloth or 
filter paper. 

Stain by dropping 10 drops of polychrome stain for blood onto 
the preparation from a dropping bottle and after one-half minute 
diluting with 20 drops of distilled water and staining two minutes. 
Float the section off by placing the slide in a second dish of water. 
After washing float the stained section onto a slide, straighten, 
drop on a square cover at the center of which a small drop of 50 
per cent, glycerine has been placed and examine. 




Fig. 60. — Surgical Specimens. Knife for cutting frozen sections. 3^- 
inch planing blade a, is set into a wooden handle b. The blades when obtained 
are usually too soft and it is necessary to have them "tempered." 

The Spencer freezing microtome (Fig. 61) (No. 880 obtained 
from the Spencer Lens Co., Buffalo, N. Y.) is widely used for cut- 
ting frozen sections and is very satisfactory. The elevation of 
the specimen by the micrometer screw is automatic. 

Not more than five minutes should be taken for the whole pro- 
cedure. The tissue left after the block has been cut for the frozen 
sections is treated as indicated under celloidin sections. 

Colloidin Sections for Routine Diagnosis — Celloidin sec- 
tions are used for the routine diagnosis of surgical specimens. By the 
celloidin method, the sections are stained, examined and reported 
on the morning following the day the specimens are received. 
This method enables a technician to do the greater part of the 
work and large numbers of specimens may be examined without 
burdensome labor or confusion. 



92 



HOSPITAL LABORATORY METHODS 




I' 



Fig. 6i. — The SpencerJFreezing Microtome. 



SURGICAL SPECIMENS 



93 



Frozen sections of formalin fixed tissue stained with hema- 
toxylin and eosin may be used for routine diagnosis, but it is 
difficult to get sections of the very small bits of tissue such as 
curettings without the use of celloidin. The celloidin sections are 
preferable to sections obtained by a rapid paraffin method. 

Care of the Tissue by the Surgeon and its Delivery at 
the Laboratory. — As soon as the tissue is removed, it is 
wrapped in sterile gauze and moistened in normal saline; oiled 
paper (i foot square) is then wrapped about the gauze (Fig. 63). 
The brown slip (Fig. 41) is folded and slipped beneath the string. 
The tissue must be sent to the laboratory as soon as possible. To 




Fig. 62. — Surgical Specimens. Microtome for celloidin sections. Bausch 
andLomb microtome No. 3026 with knife No. 3106. Slant the knife a so that the entire 
length of the edge is utilized in cutting off a section, b, 12 microns in thickness. 
80 per cent, alcohol slowly drops onto the block from bottle (Fig. 71). A number 
of sections are rapidly cut by drawing the knife back and forth. Transfer the 
sections from the knife to an Esmarch dish of water with a teasing needle. One 
of the best sections is picked out and run through the stains (Fig. 72). 



secure perfect fixation of tissue, such as is required for making 
differential stains and for making sections for class work, the tissue 
must be placed in the fixing solution within five minutes after its 
removal. 

Assignment of a Number to the Specimen. — When received 
at the laboratory, a specimen is unwrapped and completely dis- 
posed of before a second one is unwrapped. The number on the tag 
at the top of the spindle (Fig. 58) is placed on the bottom of the 
brown slip and on the sheet (Fig. 64) of a pad which has stamped 



94 



HOSPITAL LABORATORY METHODS 



on it the number, the fixation and the museum number if a museum 
specimen is made. The gross description of the specimen is 
written on this sheet. Then both brown slip and sheet are placed 
in a clip (Fig. 65) until the diagnosis has been made. The diag- 
nosis is written on the brown slip, when both ar egiven to a tech- 
nician who makes out the card (Fig. 68) for the laboratory record. 
The cards are kept in a card index case (Fig. 66). The brown slip 
with diagnosis written on it is at once returned to the clinician. 



- 










^mh 








) J x> - Q o . P • • 




c 








( qf. '" •- \ ° ■ "nq^A 


........ 


t 




\vkjj„:__ 




a 











Fig. 63. — Surgical Specimens. Surgical specimen wrapped in gauze and 
oiled paper, a, Specimen; b, quadruple layer of surgical gauze 8 inches square 
and moistened with sterile saline; c, medium thickness oiled paper 12 inches 
square. The moist gauze is wrapped about the specimen and the oiled paper 
is then used to tightly wrap the whole. Tie with ordinary twine and insert brown 
slip beneath twine. 



Fixation or the Specimens. — If a frozen section for im- 
mediate diagnosis is not asked for the tissue is unwrapped, ex- 
amined, and the gross characteristics tabulated. If the specimen 
is uterine curettings or a bit of other tissue only large enough for 
a single block, all is placed in the formalin-alcohol bottle (Fig. 
69), and the tag from the spindle placed on it. In this case the 
Z on the tag is crossed off and formalin-alcohol substituted. 
Curettings are held together by placing them with forceps on a 
piece of filter paper 1 cm. square. 

If there is enough of the tissue, pieces are also placed in a 



SURGICAL SPECIMENS 95 



S-16- Z F A M 



Fig. 64. — Surgical Specimens. Blank sheet for gross descriptions. Tablet 
paper 5 by 8 inches on which the year number (e.g., S-16-) is stamped in the upper 
left-hand corner and the three common fixatives in the upper right-hand corner. 
If the specimen is a museum specimen place number after M. 



96 HOSPITAL LABORATORY METHODS 

bottle of Zenker's fluid, unless it is an appendix, leiomyoma, or 
tube that is known to be of no interest. 

The pieces for Zenker fixation are cut after the pieces for diag- 
nosis have been placed in the formalin-alcohol and the Zenker bottle . 
receives the original number tag (Fig. 58). The Zenker-fixed 
tissue is set aside and put to wash after twenty-four hours. If it 
appears that a fat stain or a stain for bacteria may be desired, fix 
also in 10 per cent, formalin. 

Technic for the Celloidin Sections.— After the tissue has 
been placed in the formalin-alcohol (Fig. 66), the further work may 



Fig. 65. — Surgical Specimens. Paper clip, a, Margin holding paper; b f spring; 
c, hole for hanging on a nail. Measures 3 inches ; 

be placed in charge of a technician. The tissue with tag is moved 
back with a pair of forceps 10 inches long as indicated in Fig. 69. 
After remaining over night in medium celloidin, the blocks of 
tissue are dipped with long forceps into thick celloidin (the thick 
is made like medium except that it has 2 ounces of celloidin to the 
500 c.c. of alcohol-ether) and placed on a fiber block (Fig. 70) 
that has the surgical number written with a lead pencil on the 
back of the block. After a minute in the air the blocks are dropped 
into a pint Mason jar, half filled with chloroform where they 
remain for one hour. They are then transferred to 80 per cent, 
alcohol and celloidin sections are cut with a sliding microtome 



SURGICAL SPECIMENS 



97 



(Fig- 57) 1 2 to 15 microns thick. Eighty per cent, alcohol is 
dropped onto the block from a large dropping bottle (Fig. 71). 
The sections are transferred with a teasing needle (Fig. 73) from 
the knife to a dish of water. The number tag is under this dish. 
To make the medium celloidin, remove 1 ounce of celloidin 
(E. Schering) from the bottle and blot off all water with filter 
paper. Place in a pint Mason jar and add 500 c.c. equal parts 
absolute alcohol and ether. Invert the jar for several days so as to 
dissolve. 

A . • 



_________ __ 



n 




Fig. 66. — Surgical Specimens. Card-index case for surgical cards. Section 
of a card-index case 42 by 16.5 by 8.5 inches, b, is, r top which is removed and re- 
placed as additional sections are added. Shaw- Walker case, Chicago and New 
York. Five sections will serve for the indexing of the surgical specimens from a 
large hospital for a number of years and five additional ones will be sufficient for 
indexing the autopsy specimens. The front card of the drawer, a } bears a label, 
Surgicals S. 15. 1 to S. 16.3 for example. From these cards a regional cross index is 
prepared recording the "P" specimens only. "P" is explained under examination 
of the celloidin sections. 



Hematoxylin Eo sine Stain tor Celloidin Sections. — 
Hematoxylin is most often used as a nuclear stain for celloidin 
sections as many of the aniline dyes are taken up by celloidin. 
Esmarch dishes (Fig. 72) are used for the solutions. The pro- 
cedure is indicated in Fig. 72. After clearing in origanum oil 
cret. the sections are mounted in colophonium-xylol (500 c.c. 
xylol to which a pure white colophonium has been added to the 
desired consistency) . 

The numbered tag is kept under the dish in which the sections 
are, being moved forward from dish to dish with the sections. 
Before the sections are placed on the slide, the slide is numbered 
(Fig. 74). This eliminates a chance mixing of sections from 
different cases. 

7 



98 



HOSPITAL LABORATORY METHODS 




Celloidin sections may be made of tissue fixed in Zenker's 
fluid. After Zenker fixation the tissue is placed directly into the 
95 per cent, alcohol and not in the formalin alcohol. After cutting, 
the sections of Zenker material are first placed in iodine (iodine 
5 gm., and 95 per cent, alcohol 500 ex.) for fifteen minutes and 
then in 95 per cent, alcohol until all iodine is removed (one hour or 
more). The sections are stained in the hema- 
toxylin for thirty minutes instead of five min- 
utes, the time required for the formalin-alcohol 
tissue. The other technic is the same as for 
the tissue fixed in the formalin-alcohol. 

Tissue fixed in 10 per cent, formalin is 
treated exactly like the formalin-alcohol fixed 
tissue except that it is placed directly into the 
95 per cent, alcohol instead of the formalin- 
alcohol. 

Examination of the Celloidin Sections. 
— These celloidin sections are examined and a 
diagnosis made if possible. In all cases on 
which a paraffin section of the Zenker-fixed 
material is desired a "P" is placed on the label. 
The technician places the bottle of the Zenker- 
fixed "P "-tissue near the paraffin series (Fig. 
78) and a bit of this tissue is cut and started 
through the series by the pathologist. The 
Zenker-fixed and the formalin-fixed material of 
all slides so marked is kept permanently. The 
Zenker-fixed material as well as any formalin- 
mixed material of sections not so marked is 
discarded. The celloidin blocks are kept for 
one week. The celloidin slides are kept for at 
least six months. The paraffin slides made 
from "P "-tissue are kept permanently. 

Paraffin Sections. — In those cases in which it is not possible 
to make a diagnosis from the celloidin section, and in cases where 
better sections are desired for study, paraffin sections of the 



/T 



Fig. 67. — Sur- 
gical Specimens. 
Tag for celloidin 
sections. Merchan- 
dise tags 1 Y± by 2 
inches. The symbol 
in the lower right- 
hand corner indicates 
alcohol-formalin fixa- 
tion, the usual one 
used for celloidin sec- 
tions. The number 
in the left hand corner 
indicates that two 
pieces of tissue are 
in the bottle. The 
tag follows the tissue 
until the section is on 
the numbered slide. 



SURGICAL SPECIMENS 99 

Zenker-fixed material are made. For many of the differential 
stains, paraffin sections are necessary. In fact celloidin sec- 
tions are used almost exclusively for diagnostic purposes alone. 
Paraffin sections of Zenker-fixed material are used for all autopsy 
specimens. 

Zenker-fluid Fixation. — Zenker's fluid (mercuric chloride 
500 gm., potassium bichromate 250 gm., sodium sulphate 100 gm. 
arid water 10,000. Add 5 per cent, acetic acid just before using) 
is the best fixative for general histologic work and the paraffin 
sections are most often made from tissue fixed in this way. All 
tissues except the most routine specimens such as appendices, tubes, 



S- Diagnosis: 



Material: Date: 



Name: Service: 



Fixation: 



History: 



Gross: 



Fig. 68. — Surgical Specimens. Card for surgical records. Card 5 by 7 inches 
with horizontal ruling. The desired printed headings are indicated. 



IOO 



HOSPITAL LABORATORY METHODS 



fibroids, etc., are fixed in Zenker's fluid. Even such tissues as 
appendices, if obtained within five minutes after removal, should 
be Zenker-fixed. The original tag (Fig. 58) is placed on the bottle 



A ^\ ft 



ft ft ft 




Fig. 69. — Surgical Specimens. Block for celloidin imbedding, a, %-inch 
board; b, %-inch board 24 by 14 inches; c, cork-stoppered bottles obtained from 
Whitall, Tatum Co., Philadelphia. The diameter of the bottom of the bottle is 
5 cm. and that of the upper part of the mouth is 32 mm. The bottles are 11.5 cm. 
high, d, formalin (1 part) — alcohol 95 per cent. (9 parts) for one to two hours; 
e, 95 per cent, alcohol for one to two hours;/, absolute alcohol for one to two hours; 
g, absolute alcohol-ether equal parts two hours; h, medium celloidin over night. 
Eight specimens may be started at one time in this block. The following morning 
all are ready to be cut by the technician. 

containing the Zenker-fixed tissue. Besides surgical specimens, 
all autopsy specimens are fixed by this method. 

For Zenker fixation, the tissue must be cut not more than 4 mm. 




Fig. 70. — Surgical Specimens. Fiber block for celloidin sections. Fiber 
blocks 30 by 20 by 15 mm. may be obtained from Bausch & Lomb Optical Co. 
The surgical number is written on these blocks with a soft pencil. To clean these 
blocks remove celloidin by setting aside and letting dry in the air. Remove pencil 
marks with a moist cloth. 

thick, and in the plane in which the paraffin sections are to be 
cut. An exception is made to this in the case of the brain, heart 
or other large organ from which a shaving may be cut to show a 



SURGICAL SPECIMENS 



101 



superficial inflammatory process. In this case blocks for paraffin 
sections are cut at right angles to the large surface. 

Zenker's fluid is made up in a large 20-liter demijohn in the 




Fig. 71. — Surgical Specimens. Alcohol dropping bottle, a, 80 per cent, 
alcohol in a 4-liter bottle provided with a glass tube; b, with cock. This tube is 
connected to the bottle by a rubber cork d. The tip of the tube c comes directly 
above the celloidin block. This is done by placing the bottle on a support at the 
desired height. 




Fig. 72. — Surgical Specimens. Esmarch dishes for staining celloidin sections. 
a, Esmarch dish 6 cm. across and 3 cm. deep containing water; b, hematoxylin (see 
Fig. 82) for five minutes; c, Czaplewsky until excess of color is removed; d, }ioo 
per cent, ammonia for one minute; e, water; /, J^ per cent, eosin (yellowish, water 
soluble) for one minute; g, q$ per cent, alcohol 1 for one minute; h, 95 per cent. 
alcohol 2 for one minute; i, origanum oil cret. for five minutes. 



c 



Fig. 73. — Surgical Specimens. Teasing needle, a. Wooden handle; b, 

steel needle. 



bottom of which there is an excess of the bichloride. For "con- 
venient use, the Zenker's fluid, 10 per cent, formalin and 80 per 
cent, alcohol, are kept in bottles (10 liters) provided with cocks 
(Fig. 76). The acetic acid is added from a burette provided for 



102 



HOSPITAL LABORATORY METHODS 



the purpose (Fig. 77). The amount of Zenker's fluid should be 
at least ten times the amount of tissue placed in it. 







V 


___ 




1 


VV-:---.--.',.;::,:-,. :,::::.■.,:-■::■-:■;■',.:: 







Fig. 74. — Surgical Specimens. Slide with surgical number. The slide as 
it comes from the technician. The cover-glass is 22 mm. square. Before mounting 
any specimens the labels with numbers are placed on the left end of the slide. Labels 
are plain with a single narrow black or blue-line border. If for any reason a paraffin 
section is to be run and the Zenker-fixed material permanently preserved a P is 
placed on the label at the time of examination. 

The tissue remains in the Zenker's fluid for twenty-four hours, 
when it is washed (Fig. 75) for twelve hours or more, and then 
covered with 80 per cent, alcohol. 




Fig. 75. — Surgical Specimens. Sink for washing specimens, a, Sink of soap- 
stone. The upper surface inclines and has shallow furrows in it. b, water cock 
which carries a short piece of rubber tubing c. This tubing reaches into the bottle 
during the washing. A small piece of wire gauze may be placed over the tops of the 
bottles to prevent the escape of bits of tissue. 

Imbedding in Paraffin. — After remaining over night in 80 
per cent, alcohol, blocks not more than 2 to 3 mm. may be cut 
and started through the paraffin series bottles as indicated in Fig. 
78. These blocks, for example from aorta or intestinal wall, 



SURGICAL SPECIMENS 



103 



must be cut so that the technician cannot mount them from the 
paraffin in a way that they will be cut in the wrong direction. 
That is, cut so that they will be placed in the boats on their flat 
side without any questioning. 

Each morning the specimen with its tag is moved back one 
bottle. Thus on the seventh day the specimen is removed from 
the chloroform saturated with paraffin and placed in paraffin 
(extra white: melting point 52°C, Bausch and Lomb) melted in 




Fig. 76. — Surgical Specimens. Zenker's fluid, 10 per cent, formalin and 
80 per cent, alcohol bottles, a, 10-liter glass bottle with an opening into which 
a rubber cork is fitted. The rubber cork carries a glass tube with b glass cock. 
A short piece of glass tubing may be placed in the rubber cork and a piece of rubber 
tubing c provided with a pinch cock d fitted onto this, e, Bent glass tubing to 
admit air. 



paraffin oven (Fig. 79), kept at a temperature below 6o°C. The 
melted paraffin is kept in Esmarch dishes. The number tag is 
placed beneath the dish that contains the specimen. The speci- 
men is placed in a dish and after about one hour it is transferred 
to a second dish. The Zenker-fixed specimens remain in the oven 
about two hours and even three hours as Zenker-fixed material 
does not shrink in the oven. The tissue is taken from the second 
Esmarch dish and placed in a paper boat (Fig. 80) made from 
ordinary paper for this purpose. This boat filled with paraffin 
which bears the tag number on one end remains in the oven for 
about fifteen minutes and is then floated on water until the paraffin 



io4 



HOSPITAL LABORATORY METHODS 



completely hardens when the tissue blocks are carefully cut apart. 
These blocks with the number tags are placed in a pill box of 
pasteboard (Fig. 81) that bears the number on one end. The 
blocks are kept permanently in these boxes. 

Skin and certain other tissues fixed in Zenker's fluid cut with 
difficulty if imbedded in the usual way. To correct this it is often 




Fig. 77. — Surgical Specimens. Burette for glacial acetic acid, a, Wooden 
burette stand; b, 25- to 50-c.c. burette; c, piece of glass tubing bent as indicated; 
d, third hole in the three-hole rubber cork over which the ringer of the left hand is 
placed while forcing the acid over into the burette; e, wide-mouth bottle 6 inches 
high containing glacial acetic acid; /, rubber bulb with a double valve. 



advantageous to imbed in paraffin according to the method given 
under Formalin Fixation. 

Decalcification or Bone. — Bone must be decalcified. Fix 
bone sawed into pieces not more than 4 mm. thick in Zenker's 
fluid, wash and transfer to 5 per cent, nitric for twenty-four hours 
(forty-eight hours is the maximum) . Wash over night. Cut 
celloidin or paraffin sections. 

Formalin Fixation. — Ten per cent, formalin (Fig. 76) (4 per 



SURGICAL SPECIMENS 



I05 



cent, formaldehyde) is used. Frozen sections are cut from tissue 
fixed in formalin for the demonstration of fat. Formalin-fixed 
tissue is also valuable for the staining of bacteria and protozoa 
present in the tissue. All important specimens are fixed in forma- 
lin as well as Zenker's fluid. 

For the staining of organisms in the tissue very thin sections 
must be cut from paraffin blocks. No shrinkage occurs in tissue 




Fig. 78. — Surgical Specimens. Block for paraffin imbedding, a, 95 per 
cent, alcohol 1; £,95 percent, alcohol 2; c, absolute alcohol 1; d, absolute alcohol 2; 
e, chloroform; /, chloroform saturated with paraffin; g, %-inch board on which is 
nailed h, a, %-inch board into which the 2%-inch holes for the bottles are bored; g, 
is 19 inches square and h 18 inches square. The glass-stoppered bottles (Eimer 
and Amend) are 4^ inches high with stopper in place and 2% 6 inches across the 
bottom. 



during fixation in formalin as happens in the case of Zenker fixa- 
tion, but during the imbedding process very serious shrinkage 
takes place unless special care is taken to prevent it. 

For this reason formalin-fixed tissue is taken from the formalin 
and placed in 80 per cent, alcohol for twenty-four hours and then in 
equal parts of absolute alcohol and cedar oil (cedar oil clearing) for 
one day when they are transferred to a second bottle of the same. 
On the third day they are placed in cedar oil, and on the fourth day 
they are placed in chloroform saturated with 52 paraffin where 
they remain over night. Transfer to melted 52 paraffin in 



io6 



HOSPITAL LABORATORY METHODS 



the oven for thirty minutes (two changes). The specimens are 
removed from the oven and blocked the same as Zenker-fixed 
tissue. The four bottles for this formalin-fixed tissue should be 
properly labeled and kept in the block used for the Zenker-fixed 
material (Fig. 78). 

Sections from such blocks may be cut and stained with hema- 
toxylin and eosin. The paraffin is removed as indicated under 




Fig. 79. — Surgical Specimens. Paraffin oven, a, Compartment in which 
5 2 paraffin (extra white) is kept in a vessel with a pouring snout; b, shelves on 
which Esmarch dishes containing paraffin are kept. Regulate at 55 . This instru- 
ment was obtained from Bausch & Lomb Optical Co. 



eosin-methylene blue stain. Stain according to the technic given 
under celloidin sections using a long dish (b, Fig. 86) until the 
95 per cent, alcohol is reached. Transfer from 95 per cent, alcohol 
to absolute alcohol and then to xylol rather than to the origanum 
oil cret. used for celloidin sections. Mount in colophonium-xylol. 
Alcohol Fixation. — If 95 per cent, alcohol or absolute alcohol 
is used, there is a shrinkage of the tissue during fixation, and it may 
be imbedded in the same way that Zenker-fixed material is im- 
bedded. For the preservation of glycogen so that it may be 



SURGICAL SPECIMENS 



107 



stained, pieces of tissue not more than 2 mm. thick are fixed in 
absolute alcohol. 

Cutting Paraffin Sections. — -The paraffin blocks whether 
obtained after Zenker's fluid, formalin or alcohol fixation are cut on 
a rotary microtome (Fig. 82). For the usual work 7-micron sec- 
tions (two clicks) are cut but it may at times be desirable to cut 
from very dense cellular tissue such as liver, 3^-micron sections 
(one click). 




Fig. 80. — Surgical Specimens. Paper box for blocking tissue. Take a rec- 
tangular piece of paper 4 by 6 inches for example and fold the ends inward at a and 
b and then to two-thirds that distance the sides at c and d. Unfold and make the 
line ae coincide with ce and then bring/ to the point c. Fold back the lip g after all 
the corners have been treated in the same fashion. The number is written on this lip. 

As the ribbon comes from the knife it is held with a small pair 
of fine-pointed forceps, removed from the knife with the forceps 
and a needle and placed in a dish of water at about 45°C. The 
ribbon is then separated at the desired points with a heated 
scalpel. The sections are floated onto clean slides (B. and L. 
extra white 3X1 medium thickness) on which albumin fixative 
has been placed with a camel's hair brush and the excess wiped off 
with clean muslin. 

To prepare the albumin fixative thoroughly beat up the whites 
of two eggs with an equal amount of glycerine, add 1 per cent, 
sodium salicylate and let stand twenty-four hours. Filter through 
filter paper. The filtrate must be clear. If it is not clear allow to 
stand several days before filtering. 



io8 



HOSPITAL LABORATORY METHODS 



For routine work it is preferable to use No. i cover-glasses 
22 mm. by 40 mm. The length of the ribbon placed on the slide 
should be this length (Fig. 83). If No. 1 cover-glasses 22 mm. 




Fig. 81. — Surgical Specimens. Pasteboard box for paraffin blocks. White 
pill box g measuring 3% inches by 2>4 inches by i% inches. The number and 
the fixative are written on one end. These boxes are kept permanently. 




_ Fig. 82. — Surgical Specimens. Rotary microtome. This instrument is ob- 
tained from the International Instrument Co., Cambridge, Mass. Obtain at least 
twelve metal disks a to which the paraffin blocks are fastened, b bears the numbers 
by which the thickness of the sections are regulated. When set at 2 for example, 
the sections are cut two clicks or 7 microns thick. This is the usual thickness for 
routine work. Obtain at least two Minot microtome knives No. 283 from E. 
Leitz, New York City. 

square are used, the ribbon is placed at the center of the slide and 
usually consists of a single section from the block. 

To fix the specimens to the slides, the slides after the sections 



SURGICAL SPECIMENS 



109 



have been placed on them are let dry for a short time and then 
stood on edge in ten-slide staining dishes (Fig. 84) which are placed 
in the paraffin oven over night. Sections from only one block of 
tissues are floated in the dish at a time and the desired number of 




Fig. 83. — Autopsy Specimens. Slide with mounted autopsy specimens and 
label. The slide as it comes from the technician. The cover-glass is Xo. 1, 22 by 
40 mm. The examiner writes the name of the organ below the number and if it 
shows an interesting lesion one, two or rarely three checks are placed on the label. 
All autopsy sections are Zenker-fixed and stained with eosin-methylene blue. 

sections are placed on slides (Fig. 74) that bear the number of the 
tissue scratched on the end of the slide by means of a diamond 
scratch. Whatever stain is to be used, the paraffin is removed 
with xylol, the xylol removed with alcohol and the alcohol with 
water. 




Fig. 84. — Surgical Specimens. Ten-slide staining 

Optical Co. 



dish. Bausch & Lomb 



Eosin-methylene Blue Stain (Mallory). — The slides are re- 
moved from the oven and covered with xylol 1 and xylol 2 (Fig. 
84) for about fifteen minutes. This removes the paraffin. The 
absolute alcohol 1 and 2 3 and the 95 per cent, alcohol 1 and 2 are 
now poured over the slides one after the other, using a small 
funnel to pour the solutions back into the bottle. Cover the 



no 



HOSPITAL LABORATORY METHODS 



sections with water. They are now ready for the stain provided 
Zenker's fluid has not been used as a fixative. 

Mercury must be removed from all Zenker-fixed tissue. The 
mercury is removed by covering the slides after the second 95 
per cent, alcohol with iodine solution (iodine 5 gm. and 95 per 




Fig. 85. — Surgical Specimens. Bottles for staining solutions. 500-c.c. glass- 
stoppered bottles 20 cm. high with stopper in [place and 8 cm. across bottom are 
obtained from Eimer and Amend, New York City, a, 95 per cent, alcohol 1; b, 
95 per cent, alcohol 2; c, 95 per cent, alcohol for removing iodine; d, absolute alco- 
hol 1; e } absolute alcohol 2;/, xylol 1; g, xylol 2; k, 25 gm. eosinand 500 c.c. distilled 
water; i, methylene blue 5 gm., potassium carbonate 5 gm. and distilled water 500 
c.c; j, phosphotungstic acid (Merck) 5 gm., hematein ammonium (Griibler) 0.25 
gm. and water 500 c.c; k, potassium permanganate 1.25 gm. and water 500 c.c; 
/, oxalic acid 25 gm., water 500 c.c; m, aniline blue (Griibler) 2.5 gm., orange G 
(Griibler) 10 gm., phosphomolybdic acid (Merck) 5 gm., water 500 c.c; n, acid 
fuchsin 2 gm., water 500 c.c; 0, hematoxylin 5 gm., thymol crystals 1 gm., saturated 
solution of ammonium alum 500 c.c; p, Czaplewsky (5 c.c hydrochloric acid sp. gr. 
1. 19, 5 gm. sodium chloride, 100 c.c. water and 95 per cent, alcohol to 2000 c.c); 
Q) }ioo P er cent, ammonia; r, 3^ per cent, eosin (Grubler's yellowish, water soluble) ; 
s, oil of origanum ere tic (Merck). 

cent, alcohol 500 c.c.) for fifteen minutes. The iodine must be 
removed by allowing the slides to remain in the 95 per cent, 
alcohol until all trace of the brown color has been removed (one 
to two hours). After removal of the iodine, the slides are covered 
with water and are ready for the stain. 



SURGICAL SPECIMENS 



III 



The eosin-methylene-blue stain is used on Zenker-fixed tissue. 
The nuclei are usually blue but may be distinctly purplish. The 
cytoplasm of most cells is pinkish but may stain a blue as is seen 
in the case of the lymphoblastic cells. Structures such as hyaline 
in liver cells that are not even visible with other stains are brought 
out with great distinctness by this stain. 

To make the stain, the water is poured from the staining dish 
(Fig. 84) and the slides covered with a 5 per cent, aqueous solu- 
tion of Griibler's yellowish, water soluble eosin (Fig. 85) over night. 
The following morning the eosin is poured back into the bottle, the 
slides washed in water and then covered with the methylene-blue 
solution (distilled water 80 c.c, and 20 c.c. of an alkaline methylene 
blue consisting of methylene blue (Grubler's B.X.) 5 gm., potassium 
carbonate 5 gm., and water 500 c.c). The dilution with water 
should be made just before pouring onto the slides. 




c d e 

Fig. 86. — Surgical Specimens. Dishes for differentiating and dehydrat- 
ing. Eosin-methylene blue sections, a, Finger bowl 4.5 inches across top con- 
taining 95 per cent, alcohol to which 1 or 2 drops of colophonium-xylol has 
been added; b } glass dish 3^ by ij£ by % inches containing absolute alcohol 1. 
This dish may be obtained from the Marquette Medical School, c, absolute alcohol 
2; d, xylol 1; e, xylol 2. 



The time that the slides are stained with the methylene blue 
depends largely on the age of this solution. If it is less than one 
week old twenty minutes may be sufficient, while if it is one month 
old forty minutes may be required. The time (average thirty min- 
utes) is best determined by removing a slide from the dish and 
washing off under the tap. If it is sufficiently stained with the 
methylene blue, the red does not show macroscopically. After 
sufficient staining with the methylene blue this stain is washed 
out of the dish under the tap and the slides allowed to remain in 
water for a few minutes until each slide can be differentiated. 



112 HOSPITAL LABORATORY METHODS 

The differentiation (Fig. 86) of the preparations is important. 
The slide is placed first in a dish of 95 per cent, alcohol, to which 
1 drop of colophonium-xylol has been added and lifted up and 
down with a needle until a faint pink appears in the prepara- 
tion, when it is transferred to absolute alcohol in an oblong dish 1 
(6, Fig. 86). As soon as the first one is differentiated a second 
slide is placed in the dish and so on until all the dishes (one 95 
per cent, alcohol, two dishes absolute alcohol and two dishes 
xylol) are made to contain a slide. The slides are mounted in 
colophonium-xylol. 

If the slides are too red it is due usually to insufficient staining 
in the methylene blue rather than to too prolonged differentiation. 

Phosphotungstic Acid-hematoxylin Stain (Mallory). — This 
stain is used on Zenker-fixed material for the demonstration of 
fibroglia, myoglia and neuroglia fibrils. It is, therefore, especially 
valuable for the differentiation of cells. The tissue should be 
fixed five minutes post-operative. 

(1) After removal of paraffin, mercury and iodine, place the 
slide in Y± per cent, potassium permanganate for twenty minutes. 
Wash with water. (2) Oxalic acid 5 per cent, for twenty minutes. 
Wash. (3) Phosphotungstic acid-hematoxylin (hematein-ammo- 
nium (Griibler) 0.25 gm., phosphotungstic acid (Merck) 5 gm., 
distilled water 500 c.c.) over night. This stain is allowed to age 
for at least one month. Wash. (4) Differentiate in 95 per cent, 
alcohol one-half to one minute. Pass through absolute alcohol 1 
and 2, xylol 1 and 2 and mount in colophonium-xylol. 

Aniline Blue Stain (Mallory) . — This stain is used on Zenker- 
fixed material for the demonstration of collagen. It is, therefore, 
especially valuable for the determination of the presence of 
fibroblasts. 

(1) After removal of the paraffin, mercury, and iodine, wash 
and place in an acid fuchsin solution (acid fuchsin, (Griibler) 2 gm., 

^his dish, measuring at the bottom inside 3^ by i}£ inches and at the top 
sH by ij^ inches, has been made up for the author by the Canton Glass Co., 
Marion, Ind., and may be obtained in gross lots from them or in small lots from 
the Marquette Medical School, Milwaukee, Wis. 



SURGICAL SPECIMENS 113 

water 500 c.c.) for twenty minutes. Wash. (2) Aniline blue 
solution (aniline blue Grtibler soluble in water 2.5 gm.), orange G 
Griibler 10 gm., 1 per cent, aqueous solution of phosphomolybdic 
acid (Merck) 500 c.c. for twenty minutes. Wash. (3) Differ- 
entiate in 95 per cent, alcohol for one-half minute and then pass 
through the absolute alcohol and xylols to colophonium-xylol. 

Elastic Fibril Stain (Verhoeff ) . — This stain is of value in the 
morphologic study of tissue, containing many elastic fibrils, such 
as the walls of blood-vessels. (1) Zenker-fixed tissue; do not 
treat with iodine. (2) Place for fifteen minutes in the stain. To 
prepare the stain, dissolve by heat 1 gm. of hematoxylin in 20 c.c. 
of absolute alcohol in a test-tube and add 8 c.c. of 10 per cent, 
ferric chloride and 16 c.c. of an iodine solution (iodine 2 gm., 
potassium iodide 4 gm., and water 100 c.c). (3) Wash in ^ per 
cent, ferric chloride until excess of stain is removed (1 minute). 

(4) 95 per cent, alcohol to remove the iodine (one hour). Wash. 

(5) Counterstain with % per cent, eosin (one-half minute). Wash. 

(6) Dehydrate in 95 per cent, alcohol (one minute) and origanum 
oil (five minutes) and mount in colophonium-xylol. 

Scharlach R Stain for Fat (Herxheimer) . — Place for three 
minutes, frozen sections cut from formalin-fixed tissue in equal 
parts of acetone (C. P.) and 80 per cent, alcohol saturated with 
Scharlach R, transfer to 80 per cent, alcohol for a few seconds, 
wash in water and counterstain with hematoxylin. 

The sections are washed in water and mounted in glycerine 
jelly. To prepare the jelly, dissolve 50 gm. gelatin in 250 c.c. 
distilled water and add 250 c.c. of glycerine and 5 c.c. phenol. 

The saturated Scharlach R solution should be kept in a 500- 
c.c. glass-stoppered bottle with an excess of the stain in the bottom 
of the bottle. To use, filter the saturated solution into an Esmarch 
dish provided with a cover. 

Hemosiderin Reaction. — The iron reaction is valuable where 
it is desirable to ascertain the nature of a pigment. (1) Frozen, 
celloidin or paraffin sections of formalin-fixed tissue. (2) Five 
hours in 2 per cent, potassium ferrocyanide in lithium carmine 
(dissolve 5 gm. of carmine in 200 c.c. of saturated aqueous solution 

8 



114 HOSPITAL LABORATORY METHODS 

of lithium carbonate and add a crystal of thymol). (3) Czap- 
lewsky for seven hours. (4) Wash in running water thirty min- 
utes and pass through 95 per cent, alcohol and origanum oil cret. 
to colophonium-xylol. 

Glycogen Stain (Best). — Rapidly growing tumors and the tis- 
sue from cases of diabetes are frequently fixed in absolute alcohol 
for the demonstration of glycogen. The pieces of tissue not more 
than 2 mm. thick are carried from the absolute alcohol to alcohol 
and ether and then to the medium celloidin (see celloidin sections) . 

(1) Stain with hematoxylin and differentiate in the usual way. 

(2) Place for five minutes in the carmine stain. To prepare the 
carmine stain, take 6 c.c. of equal parts of methyl alcohol and 
strong ammonia and add 2 c.c. of the following solution: Add 
carmine 2 gm., potassium carbonate 1 gm., and potassium chloride 
5 gm., to distilled water 60 c.c. and heat to boiling for a few min- 
utes. Cool and add 20 c.c. of strong ammonia. (3) Differenti- 
ate for ten seconds in absolute alcohol 80 c.c, methyl alcohol 
40 c.c. and distilled water 100 c.c. Then pass through 95 per 
cent, alcohol (one minute) and origanum oil (five minutes) to 
colophonium-xylol. 

Calcium Reaction. — The calcium of the older deposits in the 
tissues is chiefly in the form of the carbonate and phosphate. 
Silver carbonate and phosphate change over to the back oxide in 
the light. 

Frozen, celloidin or paraffin sections of formalin-fixed tissue 
are used. The sections are placed in 2 per cent, silver nitrate over 
night, stained with hematoxylin, dehydrated and mounted ac- 
cording to the kind of section. After removing from the silver solu- 
tion the section, if a frozen one of formalin-fixed tissue, may be 
stained for fat by the Scharlach R method. In this case it is 
mounted in glycerine jelly. 

Gram-Weigert Stain. — This stain is fairly satisfactory for the 
demonstration of Gram-positive bacteria in sections. Thin par- 
affin sections of formalin-fixed tissue are best. (1) Hematoxylin- 
eosin stain, staining heavier than usual with eosin. (2) Gram's 
staining solution (see Bacteriological Specimens) five minutes. 



SURGICAL SPECIMENS 115 

(3) Iodine, one minute. (4) Aniline oil for fifteen minutes or 
more. (5) Xylol 1 and 2 and mount in colophonium-xylol. 

McJunkin's Polychrome Stain for Bacteria and Pro- 
tozoa. — This stain serves for the demonstration of Gram-negative 
bacteria as well as those staining by the Gram method. It has 
the additional advantage that the organisms are stained heavily. 

One drop of McJunkin's polychrome stain for protozoa is added 
to the cubic centimeter of distilled water in an oblong staining dish 
(6, Fig. 86) and one end of the inverted slide allowed to rest on 
the end of the dish (Fig. 84). This is done so as to bring the 




Fig. 87. — Surgical Specimens. Polychrome staining of slides. _ Dish 3^ by 
X M by % in. 10 c.c. of stain is placed in this dish and mixed with the lower 
end of the slide while the other end of the slide is drawn up onto the end of the 
dish so that the under preparation side is in contact with the stain. 

preparation as near to the surface of the stain as possible. The 
sections formalin-fixed and 3.5 microns thick (one click of the 
microtome, Fig. 82) are allowed to remain over night in the 
stain at room temperature or in the incubator. In winter the 
staining should always be carried out in the incubator. Wash 
in water and wipe the excess of water from about the preparations 
with cloth or filter paper. The macroscopic particles of water 
are removed from the preparations by agitating them in several 
changes of xylol (pure) containing 5 per cent, absolute alcohol 
and 1 per cent, colophonium (white). Allow the slides to remain 
in this solution from three to six hours after all visible water has 
been removed, when they are passed through two changes of xylol 
(pure) and mounted in colophonium-xylol. If there is any excess 
of blue in the sections, this is removed by placing them in the 
sunlight until the proper differentiation is secured. 

Corrosive alcohol may be used for fixation instead of formalin. 
This consists of a saturated solution of mercuric chloride two parts 
and absolute alcohol one part. 



Il6 HOSPITAL LABORATORY METHODS 

Stain for Treponema Pallidum in the Tissue Levaditi\ — 
Formalin-fixed tissue cut into thin pieces not to exceed 2 mm. in 

thickness is placed in distilled water two changes) for two days. 
Transfer to 2 per cent, silver nitrate in a glass-stoppered brown 
bottle for five days. Wash and place in a bottle containing pyro- 
gallic acid 2 gm.. formalin 5 c.c. and distilled water 100 c.c. for 

two days. Imbed in the usual way in paraffin, cut sections, 
remove the paraffin with xylol and mount in colophonium-xylol. 

Stain for Tubercle Bacilli in the Tissue. — It is )ften re- 
quired to stain for tubercle bacilli in the celloidin sections used 
for diagnosis. A section as thin as can be obtained is floated 
onto a slide, the excess of water removed with a niter paper and 
absolute alcohol run onto it from a dropping bottle until com- 
pletely dehydrated. It is then smoothed out by pressing firmly 
with filter paper and the slide is placed for five minutes in a 4 
by ii-cni. stoppered bottle with 1 cm. of ether in the bottom. 
Remove and drop 95 per cent, alcohol on the preparation without 
permitting it to dry and then wash with water. Stain lightly with 
hematoxylin, wash with water and stain with carbol-fuchsin see 
Ziehl-Neelsen stain for three minutes, steaming over a water- 
c;-:h or gently over a flame. Decolorize with Czaplewsky until 
the section is only faintly pink not more than thirty seconds ) and 
then place in 1 5 r,ri per cent, ammoni- for a few minutes. Dehy- 
drate rapidly in 95 per cent, alcohol (thirty seconds and then in 
origanum oe five minutes . Mount in colophonium xylol. 

Paraffin sections of formalin, alcohol or Zenker-fixed material 
are stained in this manner except that the sections arc fastened 
to the slide by placing in the paraffin oven in the usual wa 
Following this fixation to the slide the paraffin is removed with 
xylol. 

Stain eor Leprosy Bacilli in the Tissue Flexner . — Cut 
thin paraffin sections of formalin-fixed tissue. 1 Alum hem- 

xylin for thirty minutes. Decolorize in Czap lewsky and trans- 
fer to 1 5r, per cent, ammonia. (2) Carbol fuchsin over night at 
room temperature. Wash. (3) Iodine (iodine 1 gm.. potassium 
iodide 2 gm. 3 and water s zz c - c - Ior one minute. Wash. (4) 



SURGICAL SPECIMENS 



II 7 



Aniline oil two hours or longer until the bacilli can be seen with 
the oil immersion. (5) Xylol to colophonium-xylol. 

Myelin-sheath Stain (Weigert). — This stain colors the fibers 
that have their myelin sheaths intact. Blocks of tissues for such 
staining should be not more than 4 mm. thick. They are cut 
through the gross structures in such a way that in the sections the 
fibers will be cut transversely. After this stain an area of degener- 
ated nerve fibers appear lightly stained when examined with the low 
power. 

The technic of the stain is ab follows: (1) Blocks less than 

4 mm. thick from formalin-fixed tissue. (2) Potassium bichromate 

5 gm., fluorchrome 2 gm., and distilled water 100 c.c. for four days. 
(3) Acetate of copper 5 gm., glacial acetic acid 2 c.c, fluorchrome 2 
gm., and distilled water 100 c.c. for one day. (4) Imbed and cut 
10 micron celloidin sections. (5) Stain the sections for one day in 
10 per cent, hematoxylin in absolute alcohol (two weeks old) 10 
c.c, saturated solution of lithium carbonate 1 c.c and water 90 c.c 
Wash. (6) Differentiate in borax 2 gm., potassium ferricyanide 
2.5 gm., and water 200 c.c Wash. (7) Dehydrate in 95 per 
cent, alcohol and origanum oil and mount in colophonium-xylol. 

Degenerated Myelin-sheath Stain (Marchi). — In distinction 
to the Weigert stain, this stain colors only the degenerated sheaths. 
Place 4-mm. blocks of formalin-fixed tissue in Muller's fluid 
(potassium bichromate 25 gm., sodium sulphate 10 gm., water 
1000 c.c.) for ten days. Transfer to Marchi's fluid (Muller's fluid 
200 c.c and 1 per cent, osmic acid 100 c.c) for ten days. Wash 
for one day in distilled water. Cut 10 micron celloidin sections. 
Dehydrate the sections in alcohol and then pass through chloro- 
form and mount in colophonium-chloroform. 

Ganglion Cell Stain (Golgi). — Place bits of the nervous tissue 
not more than 4 mm. thick in 2 per cent, potassium bichromate 
for six weeks and then transfer to Jl2 per cent, bichloride of 
mercury for six weeks. Dehydrate in absolute alcohol for fifteen 
Tninutes and place in medium celloidin for twenty-four hours. 
Cut thick sections. The sections are dehydrated for one minute 
in 95 per cent, alcohol, transferred to origanum oil cret. (five 
minutes) and then to colophonium-xylol. 



Il8 HOSPITAL LABORATORY METHODS 

Chromaffin Cell Stain. — Fix in a saturated solution of potas- 
sium bichromate (dissolve 10 gm. potassium bichromate in ioo c.c. 
water with heat, cool and filter) for twenty-four hours, wash over 
night in running water, cut paraffin sections and stain with 
Mallory's eosin-methylene blue stain. 

AUTOPSY SPECIMENS 

Introduction. — Surgical specimens may be fixed one minute 
post-operative and are the best for fine histological work. On the 
other hand, the possibility of examination at autopsy of all the 
organs has the advantage of showing the relationship and the 
extent of the disease processes present. An effort should always 
be made to hold the post-mortem examination as soon after death 
as possible. If the body is at once placed in a morgue cooled by 
an efficient refrigerating apparatus, a period of three to six hours 
between the death of the individual and the autopsy does not 
affect so seriously the preservation of the tissue. Material from 
autopsies one hour post-mortem is satisfactory for microscopic 
examination. 

The autopsy room is provided with a zinc, copper or soap- 
stone autopsy table equipped with running water. A board 
3 ft. square on which to place instruments and another on which 
to section organs are provided. Scales with a full set of weights 
and large scoop pans, a dish with Zenker's fluid and a pint Mason 
jar containing 10 per cent, formalin are placed on a small table 
which is drawn up against the foot of the autopsy table. A 
Bunsen flame, an inch square disk of iron with a handle set in wood 
for cauterizing, a scalpel to be flame-sterilized, a platinum loop and 
culture media are also placed on this table. Suitable instruments 
(Fig. 88) are important. The size and character of these instru- 
ments are given in the figure. A printed form-sheet (Fig. 92) 
is used for taking down the essential data during the autopsy. 

The body is placed on its back on the table and a block of wood 
(Fig. 89) hollowed out so as to support the head is placed beneath 
the neck. The operator stands at the right side of the subject. 



AUTOPSY SPECIMENS 



II 9 



The data, such as the time post-mortem, edema, etc., are deter- 
mined and recorded as indicated on the slip. A brief clinical 
history and the probable clinical diagnosis are obtained either 
from a written record supplied by the clinician or rom the clinician 
present at the autopsy. 




Fig. 88. — Autopsy Specimens. Autopsy instruments. Obtained from Codman 
and Schurtleff, 120 Boylston St., Boston, a, Cartilage knife with blade 4 by 11 cm.; 
b, cartilage knife with blade 3 by 9 cm.; c, brain knife with blade 2 by 24 cm.; d, 
saw with blade 4 by 25 cm.; e, hatchet chisel with a blade 5 by 5 cm.;/, metal mallet; 
g, angular bone forceps 45 cm. long; h, enterotome 21 cm. long; i, head holder, a 12- 
cm. blade; /, mouse-tooth forceps 20 cm. long; k, broad ligament clamp with a 12- 
cm. blade; /, costotome 23 cm. long; m, scissors 12 cm. long; n } curved cutting needle 
10 cm. long; o, probe 35 cm. long. 

External Body Surfaces. — Besides the determination of devel- 
opment, nourishment, edema, etc., already noted, a more careful 
external examination is made. Positive findings alone are re- 
corded except when there is a special reason to record a negative 
one. 

The external examination should begin with the scalp where 



120 



HOSPITAL LABORATORY METHODS 



wounds, bruises and scars are looked for. The mouth should be 
inspected for tumors and inflammatory processes and for abnormal 
pigmentation. 

The neck is examined for enlargements in connection with the 
salivary glands, thyroid gland and lymph nodes. Asymmetry 
in the thorax may be important. 

The abdomen may be distended with gas (usually within the 
intestines) or fluid. Large veins may appear superficially in liver 
cirrhoses. Tumor nodules may appear in the abdominal skin as 
well as on the skin surface elsewhere. Laparotomy wounds are 




Fig. 89. — Autopsy Specimens. Block of wood for supporting wood. Wooden 
block 12 by 5 by 5 inches with the groove 6 inches across and 3 inches deep. 

noted. The external genitals are examined for discharges and old 
scars. The extremities are examined for abnormalities in size 
and shape. Tattoo markings on the arms and chest may aid in 
identification. 

Peritoneal Cavity. — An incision (Fig. 90) is made down the 
median line of the body from a point just above the center of the 
sternum to the symphysis pubis avoiding the umbilicus. This 
incision is carried down to the sternum but on the abdomen it 
extends only through the skin and subcutaneous fat. After 
completing the main incision, nick into the peritoneal cavity just 
below the sternum and insert two fingers of the left hand and cut 
down between these fingers to the symphysis. 

Cut the peritoneum and muscles off from the ribs and cartilages 



AUTOPSY SPECIMENS 



121 



below the thorax and with long sweeps of the cartilage knife 
strip all the thoracic muscles back with the skin (Fig. 90). Ex- 
amine the intercostal muscles for trichinella infection. Examine 
the mammary gland by cutting into it from the muscle surface. 

The viscera contained in the peritoneal cavity are examined. 
Free fluid is looked for especially in the pelvis. The position of 
the lower border of the liver and 
of the diaphragm on the two sides 
is noted. The appendix, the gall- 
bladder and the general peritoneal 
surfaces are examined for evidence 
of inflammation and tumor. Ex- 
amine the organs contained in the 
pelvis. 

Pleural Cavities. — With the 
costotome the ribs are cut through 
from below upward just within 
the nipple line (Fig. 90), converg- 
ing toward the costo-chondral 
junction above until the first rib 
is reached. The first rib should be 
cut through not at the costo- 
chondral junction but outside the 
mid-clavicular line. This is done 
by angling the costotome so as to 
bring the upper blade beneath the 
clavicle, when it is pushed through 
between the clavicle and rib and the rib cut. 

Now pull the lower end of the sternum upward and with the 
cartilage knife dissect off the soft tissues from its under surface 
and cut through the tissues along the lines where the ribs are cut. 
The sterno-clavicular joint is disarticulated from beneath and the 
sternum removed. Examine the mediastinal structures for tumor, 
aneurism and enlarged thymus. The two pleural cavities are in- 
spected; fluid, inflammatory processes, old fibrous adhesions and 
metastatic tumor nodules are looked for. 




Fig. 90. — Autopsy Specimens. 
Main autopsy incision. Incision 
extends from an inch above the 
sternum a to the pubis b. Strip 
back the thoracic muscles and cut 
along the lower border of the ribs 
at c. The ribs are cut along the 
line d with the costotome. 



122 HOSPITAL LABORATORY METHODS 

Pericardial Cavity. — The position of the apex is noted before 
the sac is opened. To open the sac catch the pericardium at its 
center with forceps and first cut upward over the great vessels 
with scissors. Maintain the hold with the forceps and continue 
the incision downward along the right side of the heart. From 
the center of this incision cut downward to the apex. 

If death is very slow there may be ioo c.c. of pericardial fluid. 
If death has not resulted from cardiac paralysis the left ventricle 
will be contracted. Note adhesions, inflammations and tumor 
nodules. 

Open the pulmonary artery in situ by cutting into it with 
sharp-pointed scissors and opening up toward the lungs. Remove 
the heart by lifting it upward with the left hand and cutting the 
vessels from below upward. 

Heart. — Weight, 275 gm. Tricuspid 12.5 cm.; pulmonary 9.5 
cm.; mitral 10.5 cm.; aortic 7.5 cm.; left ventricular wall 1 cm. 

To open the heart, it is placed on a board with the anterior 
side up. With the enterotome open the right auricle by cutting 
between the openings of the inferior and superior vena cava. Ex- 
amine the auricular appendix for thrombi. 

Now with one cut of the enterotome open through the tricuspid 
valve and down the extreme right side of the right ventricle to its 
apex. From the middle of this incision push the enterotome 
through the pulmonary orifice and lay it open with one cut. 

After examining the mitral orifice from above, push the flat 
blade through the orifice and open the left ventricle along its left 
border to its apex. The short blade of the enterotome is inserted 
into the cavities except in opening the aortic and the pulmonary 
orifices. From the apex of the left ventricle cut up along the 
ventricular septum and with one cut lay open the aorta. 

Lungs. — Free the lungs by breaking any adhesions that may 
be present. If the adhesions are very firm, it may be necessary 
to cut the diaphragm off with the base of a lung. Lift the left 
lung upward by allowing the root to pull through between the 
first and second finger and cut through the root from above down- 
ward. Right and left lungs are removed in the same way. 



AUTOPSY SPECIMENS 



123 



Open the vein, artery and bronchus to some distance with 
probe-pointed scissors. Incise the lung with one long sweeping 
cut extending from the pleural surface down to the root (Fig. 91), 
the anterior edge of the lung being held in the left hand with a 
finger between the lobes. 

Spleen.— Weight, 175 gm. Incise the vessels near the hilus and 
then pull the organ from its position beneath the diaphragm. Ex- 
amine the capsular surface for a 
infarcts, fibrous thickenings, in- 
flammations, and tumors. Note 
its consistency. Place on the 
board and incise in its long 
diameter and examine the cut 
surface for the normal lymph 
nodules and for abnormal collec- 
tions of cells, such as typhoid 
nodules. Now make numerous 
cuts in the short diameter. 

Gastrointestinal Tract. — 
Cut the omentum off near the 
transverse colon. Draw the 
sigmoid colon forcibly upward 
and with the knife free it from 
its mesocolon down to the rectum where a double ligature or 
two clamps are placed on the gut. Cut between the ligatures 
or the clamps. Free the transverse colon by cutting the lesser 
omentum connecting it with the stomach. Pull loose the 
ascending colon and cecum and then by pulling steadily so as 
to put the mesentery of the small intestine on the stretch cut it 
off near the gut by a sawing motion of the brain knife until the 
duodenum is reached. With the scissors cut off the mesentery as 
near its attachment as possible. 

Double clamp the lower part of the duodenum and cut. Sepa- 
rate the stomach from the liver by cutting the hepato-duodenal 
ligament, noting the contents of the portal vein and hepatic artery 
and probing the common bile duct. Now cut through the dia- 




Fig. 91. — Autopsy Specimens. Lung 
incision. Right lung, a, Apex; b, thin 
margin; c, fissure in which thumb is 
placed; d, long sweeping incision made 
with brain knife. 



124 HOSPITAL LABORATORY METHODS 

phragm when the duodenum, stomach and pancreas are removed 
by dissecting upward beneath these structures. Clamp the esopha- 
gus low down and cut. 

Remove the intestines to the sink and with the enterotome 
open the colon along one of its longitudinal muscle bands and the 
small intestine along the attachment of the mesentery, examining 
the entire mucous surface carefully. Open the stomach along the 
lesser curvature and continue the incision through the duodenum 
using small scissors. 

Pancreas. — Weight, ioo gm. Cut the stomach off above and 
place the pancreas on the board with the duodenum to examine 
the ducts. Numerous cross-sections are made through the organ. 
The islets are more numerous in the tail. 

Liver. — Weight, 1800 gm. Make a deep incision transversely 
through both lobes, place the thumb in this incision and pull up- 
ward and cut through all attachments, first those of the right lobe 
and then those of the left. 

Examine the capsule for irregularities and the cut surface to 
determine the condition of the lobules. The markings that cor- 
respond to the periportal tissue anastomose, while the areas that 
correspond to the central portions of the lobules do not. This is 
important as the color of these structures varies greatly, and in 
different pathologic processes the same structures appear differ- 
ently. Make cross-incisions at right angles to the first cut. 

Kidneys. — Weight of the two kidneys, 300 gm. Make an in- 
cision through the peritoneum and the perinephritic fat along the 
outer border of the kidney which is then grasped and pulled out 
of its fatty capsule. Cut the ureter several inches below the pelvis. 

Place the kidney in the palm of the left hand and with one 
sweep of the brain knife cut through to the hilus from the convex 
border. Strip the fibrous capsule back on either side with forceps. 
Note the ease with which the capsule strips and the appearance 
of the kidney surface beneath it. Note the thickness of the cortex 
at its narrowest point above the pyramids (4 mm.), the appearance 
of the glomeruli, the straight tubules of the cortex (cortical rays) 
and the convoluted tubules between these (labyrinth). 



AUTOPSY SPECIMENS 1 25 

Adrenals. — The adrenals are removed with the fat about them 
and then dissected free from the fat. The right one lies close up 
against the liver. ■ Incise transversely and note the white center 
(medulla), the brown zone next this (pigmented reticularis) and 
the outer yellow part (two outer zones of the cortex). 

Genito-urinary Organs. — Strip the peritoneum from the pelvic 
walls beginning in front of. the bladder. Continue to strip down 
to the lower end of the rectum which is cut through with the knife. 
Cut through the urethra in front of the prostate in the male and 
through the vagina in the female and remove the pelvic organs in 
toio. 

Open the rectum posteriorly and wash. Open the bladder by 
cutting up through the urethra and its anterior wall. 

Incise the vagina through the bladder wall and continue the 
incision up through the anterior wall of the uterus and then 
laterally extend it out through the tubes. Incise the ovaries in the 
long diameter. 

To examine the testicles cut through the tissues beneath the 
skin on either side of the root of the penis and push the testicles 
up through the incision. Incise them in the long diameter. 

Aorta. — Open the aorta anteriorly along its entire extent and 
continue the incision down the iliacs. 

Neck Organs. — Continue the main incision at least i inch 
above the sternum and if permissible up to the chin. With the 
smaller cartilage knife dissect up beneath the skin in front and then 
behind, keeping against the vertebrae. Allow the head to drop back 
over the block and run the brain knife up in front of the larynx 
until the point appears in front of the tongue. Turn the knife and 
with a sawing motion cut back along the ramus of the jaw on 
either side until the posterior wall of the pharynx is reached which 
is divided as high as possible. During the entire operation, with 
the left hand make strong traction on the trachea and esophagus 
from below. Now pass the left hand up and pull the tongue strongly 
downward and cut the remaining attachments on either side and 
remove the organs in one mass. 

Take care to cut outside the tonsils. 



126 HOSPITAL LABORATORY METHODS 

On the anterior surface, incise each lobe of the thyroid in its 
longest diameter. Examine the pharynx and incise the tonsils. 
Now turn the mass over and slit the esophagus down in the 
median line posteriorly. Slit the larynx and trachea down in 
the median line posteriorly. 

Brain. — Insert a sharp-pointed scalpel at the hair margin just 
behind the right ear and cutting outward, carry the incision 
over the vertex of the skull to the same point behind the left 
ear. Strip back the anterior and posterior flaps by grasping with 
the hands and cutting only when necessary. Place the hair if long 
beneath the flaps and wrap a towel about the neck to protect it 
from blood. 

Outline the line to be sawed by cutting through the periosteum 
and temporal muscle. This line extends from the ends of the 
scalp incision straight around the forehead over the frontal emi- 
nences. Posteriorly two straight lines are carried back to a 
point just in front of the occipital protuberance. 

The head holder is clamped on and screwed down firmly with 
the clamps in the temporal region. Saw down to and partially 
through the inner table along the lines marked. Drive in the 
chisel with the hammer to crack through the inner table. With 
the chisel inserted in the frontal region pry off the calvarium. 

Inspect the dura and then cut through it with scissors along 
the lines sawed and strip back by cutting the falx cerebri where 
necessary. Lift up the brain with the left hand and cut the optic 
nerves, other cranial nerves and the carotids. Draw forward the 
temporal lobes and cut the tentorium with knife. Cut the cord 
as low down as possible and remove the brain. 

Examine the vessels at the base following the middle cerebrals 
into the fissures of Sylvius. Place the brain on the board with 
base down. Spread the two cerebral hemispheres apart and cut 
through the corpus callosum on each side and extend the incisions 
into the anterior and posterior cornua, exposing the lateral 
ventricles and the basal ganglia. Now make two incisions along 
these cuts with the brain knife carrying them down through^the 
basal ganglia. Cut the corpus callosum in front and reflect back- 



AUTOPSY SPECIMENS 1 27 

ward. By drawing back the velum interpositum the third ven- 
tricle is exposed. Outside these two primary longitudinal incisions 
through the brain substances make two more on each side parallel 
to the first ones. The cerebellum is cut through in the median 
line and then two incisions made through each of the halves. 

Middle Ear. — With the heavy bone forceps bite off the roof of 
the middle ear (petrous portion of the temporal bone). 

Naso-pharynx (Harke). — Extend the original scalp incision 
behind the ears downward to the middle of the neck. Dissect 
the posterior flap down and uncover the upper cervical vertebra. 
Dissect the anterior flap down and uncover the root of the nose 
and the upper edge of the orbits. Saw down through the base of 
the skull in the antero-posterior median line, going slightly to one 
side through the frontal bone so as to avoid the nasal septum. 
Cut the ligaments attached about the foramen magnum and 
forcibly push the two halves of the skull apart. Examine the sphe- 
noidal sinus, the frontal sinus, the nasal passage and the antrum of 
Highmore. 

Bone-marrow. — Make a long incision over the middle of the 
thigh, dissect and push the muscles aside so as to expose the middle 
third of the femur. Saw into the marrow cavity of the femur with 
two incisions separated on the surface of the bone by 3 cm. and 
in the cavity by 1 cm. With the. bone forceps split out this chip 
of bone, exposing the marrow. 

Spinal Cord. — Place the body face down with a block under the 
chest. Make a median line incision extending the entire length 
.of the spinal column and strip all the soft tissue back on the two 
sides. Saw through the laminae and bite off the spinous processes 
with heavy bone forceps. The arches of the cervical vertebrae 
are bitten off with the bone forceps. Divide the nerve roots with 
a scalpel. Remove the cord with dura intact. Slit the dura open 
posteriorly and anteriorly. Make transverse cuts at a distance of 
1 cm. 

Fixation of Autopsy Specimens. — As a routine all organs are 
fixed in Zenker's fluid and in 10 per cent, formalin. The Zenker's 
fluid (usually 2 liters) is placed in a flat dish on the table. At 



128 



HOSPITAL LABORATORY METHODS 



AUTOPSY SHEET FOR TEMPORARY RECORD. 



Autopsy No. 
Name: 
Nourish. : 
Edema: 

Brief clinical history: 

Clinical diagnosis: 
External body surfaces: 
Peritoneal cavity: 
Pleural cavities: 
Pericardial cavity: 
Heart weight: 
Lungs weight: 
Spleen weight: 
Gastro-intestinal : 

Pancreas: 
Liver weight: 
Kidney weight: 
Adrenals: 

Genito-urinary organs: 
Aorta: 

Neck organs: 
Brain weight: 
Middle ear: 
Naso-pharynx: 
Bone-marrow: 
Spinal cord: 

Anatomical diagnosis: 



Hours p.m. Age: B.L. 

Date: Sex: Color: Develop.: 

Rigor mortis: Lividity: 



Fig. 92. — Autopsy Specimens. Autopsy sheet for temporary record. Such 
sheets should be 8)4 by 16 inches. 



AUTOPSY SPECIMENS I 29 

completion of the autopsy this dish is covered and after twenty- 
four hours placed to wash. After washing over night the speci- 
mens in the dish are covered with 80 per cent, alcohol. 

Blocks for paraffin sections are now cut from each organ and 
placed in a small bottle of 80 per cent, alcohol with tag attached 
on which is indicated the autopsy number, the number of blocks, 
and the fixation. The bottle is given to a technician who runs the 
tissue through the paraffin series (Fig. 78) and cuts sections 7- 
microns thick which are stained with eosin-methylene blue and 
mounted in colophonium-xylol. 



Fig. 93. — Autopsy Specimens. Temporary autopsy file, a, Pasteboard for 
protection of autopsy sheets; b, board 1 6 J^ by 9^ inches; c 3 holes punched in autopsy 
sheets by a paper punch of the proper width. Obtained from any first-class stationer. 

Cover-glasses No. 1,22 X 40 mm. are best for autopsy work, so 
that several sections may be placed on each slide. The slide bears 
the autopsy number scratched on it with a diamond scratch. 
After mounting, * plain white labels with narrow black border 
are placed on the left end of the slides with the autopsy number 
above. 

The slides are placed in a flat tray and the examiner places 
the name of the organ on each. The sections showing interesting 
lesions are checked. 

The formalin-fixed tissue is used especially for frozen section 
for a fat stain. The pint Mason jar containing the formalin-fixed 
tissue is kept for one year and the one containing the Zenker-fixed 
material in 80 per cent, alcohol is kept permanently. 



130 HOSPITAL LABORATORY METHODS 

Cutting the tissue from the organs for fixation in the Zenker's 
fluid is extremely important. Usually it is best to fix tissue at the 
time the organ is examined. From the large organs cut blocks of 
tissue several centimeters in size, place these firmly on the board 
and with a sharp brain knife cut thin slices of tissue not to exceed 
4 mm. in thickness. Select the part to be fixed and in general 
cut the section in the same direction that the microscopic sections 
are to be cut. However, in the case of intestine and other thin- 
walled organs a portion of the wall may be sufficiently thin to 
fix properly. Include the surface of the organ where possible. 
Fix an abundance of any organ that shows a macroscopic lesion, 
especially if the autopsy is fresh. 

The autopsies are given consecutive numbers, for example, 
A 16.101 is the 101st autopsy made in 1916. This method of 
numbering is explained under surgical specimens. The number and 
fixation is indicated on a merchandise tag which is attached to the 
wire clamp of the Mason jar. As soon after the autopsy as 
possible the operator takes the autopsy sheet (Fig. 92) and 
from this dictates the autopsy protocol to a stenographer. How- 
ever, the stenographer may take the dictation during the per- 
formance of the autopsy. 

The protocol is typewritten on paper measuring 8% X 13 
inches, plain except for one vertical ruling at the left margin. 
The records for the current year are kept on this file after which 
they are bound. 

The bacteriological findings are added from a bacteriological 
card that bears the autopsy number. The microscopic findings 
are dictated for addition to the protocol at which time changes 
and additions in the anatomic diagnoses are made. 

At the time of the autopsy a card is made out with the name 
of patient, age, sex, service and clinical diagnosis. The number 
of autopsy is ascertained from these cards. Later the corrected 
anatomic diagnoses are added to the card. 

Museum Preparations (Fig. 94). — It may be required to 
make museum preparation from either surgical or autopsy speci- 
mens. In either case the tissue or organ is given a number (for 



AUTOPSY SPECIMENS 



131 



example, M 7650 is the 7650th museum specimen made in the 
laboratory). 

If a surgical specimen, brief gross description is written on the 
blank sheet (Fig. 64) that goes with the brown slip to the technician, 
and from this the museum card bearing not only the museum number 
but also the surgical or autopsy number is at once made out. 

If an autopsy specimen, a brief gross description is taken from 
the temporary autopsy sheet and from this the museum card is at 
once made out. There is a spindle on which muslin slips 1X4 




M 4-80 




Fig. 94. — Autopsy Specimens. Museum preparations, a, Round museum 
jars in which a heart or other specimen that requires removal for demonstration is 
placed; b, rubber band; c, screw for tightening the cover; d, the museum number 
consisting of a plain letter and number. The letters and numbers are obtained from 
The Tablet and Ticket Co., 624 W. Adams St., Chicago, e, Rectangular museum 
jars in which the specimens are sealed. Both round and rectangular jars may be 
obtained from Eimer and Amend, New York City. 

cm. are kept. These bear the consecutive museum numbers so 
that the number for a new specimen may be ascertained either 
from this tag file or from the cards. At the time of making the 
preparation the surgical or autopsy number, as the case may be, 
is also placed on the muslin slip. 

After attaching the numbered muslin slip to the specimen by 
means of a large cutting needle and coarse thread, the specimen 
trimmed and supported in such a way as to show best the lesion 
present, is placed for several days in a large 5- to 10-gallon crock 
containing J^ P er cent, concentrated ammonia in 10 per cent. 



132 HOSPITAL LABORATORY METHODS 

formalin. Transfer to 95 per cent, alcohol containing 10 per cent, 
glycerine until the desired color is secured (about two hours). 
Place in a third jar containing Russian mineral oil number 2. 
After a week or more in this jar place in oil in suitable museum 
jars (Fig. 94) with the museum number placed on top or sides 
with mucilage letters and numbers. These letters of the size 
desired may be obtained from the Tablet and Ticket Co., 624 
W. Adams St., Chicago, 111. 

The jars are sealed with a combination wax consisting of 300 
gm. bee's wax, 100 gm. rosin and 100 gm. soft black rubber tub- 
ing melted up in a porcelain dish in which the wax is kept perma- 
nently. The wax is melted and applied to the surface to be sealed 
with a camel's hair brush. A flame from a Bunsen burner may 
be used to melt any irregularities. 

Hand Lotion (Mallory). — In handling museum specimens 
avoid getting formalin on the hands. If the hands become rough- 
ened a hand lotion may be used. This lotion is prepared by 
placing 1000 c.c. of distilled water, 50 c.c. U. S. P. glycerine, 
20 gm. boric acid and 15 gm. (unpowdered) gum tragacanth (the 
amount varies with the quality) in a bottle and shaking for four 
days. Then filter through a towel onto which 1 c.c. of oil of 
geranium has been poured. A good grade of tragacanth should 
be used. 



INDEX 



Abdomen, opening of at autopsy, 120 
Acid alcohol, 81 

Adrenals, removal of at autopsy, 125 
Agar, ascitic, 75 

blood, 75 

glucose, 75 

glycerine, 75 

plain, 73 
Agglutination, 50 
Albumin fixative, 107 
Albumin in cerebro-spinal fluid, 83 

in urine, 1 

alcohol fixation, 106 
Alkaline methylene blue, 1 1 1 
Alum carmine, 114 
Alum-hematoxylin, 97 
Amboceptor, 58 
Anerobes, cultivation of, 84 
Animal autopsy board, 82 
Animals, inoculation of, 83 
Antigen, 59 
Autopsy, instruments, 119 

method of section, 120 

preparation of body for, 118 

records, 118 

Bacillus aerogenes capsulatus, 84 

diphtherias, 85 

dysenteriae, 24 

leprae, 116 

tetani, 84 

tuberculosis, 83, 24 

typhosus, 24 
Bacteria, anerobic, cultivation of, 84 

capsules of, 82 

cultivation of, 72 

demonstration of in tissue, 115 

Gram's stain for, 79 



Bacteria, spores of, 82 

Bacterial vaccines, 87 

Balsam, 97 

Best's stain, 114 

Bile pigment in urine, 8 

Blackening of wood-work, 89 

Blood, chemical test for, 24 

coagulation time, 40 

corpuscles, counting of, 37 

culture of, 52 

defibrination of, 58 

differential counting of leuco- 
cytes, 42 

endothelial leucocytes, 47 

hemocytometer, 37 

hemoglobin estimation, 33 

leucocytes, counting of, 42 

lymphoblastic cells, 43 

Mcjunkin's polychrome stain for, 
50 

Mcjunkin's tube for cultures, 

53 

myeloblasts cells, 46 

phagocytosis in vitro, 50 

platelets, 48 

protozoa in, 48 

serum for Widal reaction, 50 

staining of films, 42 

typhoid bacilli in, 54 

Wassermann test, 55 

Widal reaction, 50 
Body, external examination of, 119 
Bone, decalcification of, 104 

saw, 119 
Bothriocephalus latus, 28 
Bottles, for autopsy specimens, 105 

for surgical specimens, 100 

snout-leak dropping bottles, 78 



133 



134 



INDEX 



Bouillon, preparation of, 75 
Brain, section of, 126 

Calcium deposits, stains for, 114 
Capsules, stains for, 82 
Carbol-fuchsin, 80 
Carbon dioxide for freezing, 89 
Carmine, lithium, 114 

Best's, 114 
Cartilage knife, 119 
Casts of bronchioles, 29 

in urine, 17 

of bronchi, 29 
Cedar- wood oil, 105 
Celloidin imbedding, 100 

microtome, 93 

sections, fiber blocks for, 100 

staining of, 97 

use of, 91 
Centrifugation of defibrinated blood, 

63 

of urine, 13 
Cerebro-spinal fluid, 83 
Chloroform for paraffin imbedding, 

105 
Chromaffin cells, stain for, 118 
Clearing, oleum origani cretici for, 

97 
Collagen, Mallory's stain for, 112 
Colonies, "]2 
Colophonium, 97 
Complement fixation, 55 
Corrosive-alcohol fixation, 115 
Costotome, 119 
Cover-glass, size of, 41 
Cover-glass forceps, Novy, 43 

blood films on, 41 

freeing from fat, 41 
Cultures, anerobic, 84 

blood, 52 

examination of, 79 

for diagnosis of diphtheria, 85 

for gas bacillus, 84 

for tetanus, 84 

for typhoid, yj 

media for, 72 

pure, 72 



Cultures, use of, y2 
Curschmann's spirals, 30 
Czaplewsky fluid, 81 

Damar, gum, for label varnish, 79 
Decalcification, 104 
Degenerated myelin sheath, 117 
Delafield's hematoxylin, no 
Dextrose agar, 75 
Diagnosis, clinical laboratory, 88 

bacteriological, 68 

pathological, 88 
Dilution; method of, 7 
Diphtheria bacillus, 85 
Dittrich's plugs, 29 
Drigalski medium, "/y 
Dropping bottle for celloidin sections, 
101 

for stains, 78 
Dunham's peptone medium, yj 
Dysentery bacillus, 24 
Dura, examination of, 126 

Ear, examination of middle ear, 

127 
Erlenmeyer flask for defibrinating 

blood, 54 
Elastic fibril stain, 113 
Elastic fibrils in sputum, 30 
Endotherlial leucocytes in blood, 47 

in sputum, 30 

in urine, 17 
Entameba histolytica, 25 
Enterotome, 119 
Eosin- methylene blue stain, Mallory's 

109 
Eosinophiles in blood, 46 

in sputum, 30 
Epithelial cells in sputum, 30 

in urine, 17 
Erythrocytes, 48 

Fat, stain for, 113 

Fatty acid crystals in feces, 23 

in sputum, 30 
Feces, ameba in, 25 
bacteria in, 24 



INDEX 



135 



Feces, blood in, 24 

flagellates in, 25 

hook-worm in, 26 

infusoria in, 26 

macroscopic examination of, 22 

method of collection of, 22 

ova in, 26 

protozoa in, 25 

tapeworm ova in, 26 

tubercle bacilli in, 24 

typhoid bacilli in, 24 

undigested food particles in, 23 

worms in, 26 
Fermentation, test for sugar, 7 
Fermentation-tube, 83 
Fibrils, collagen, 112 

elastic, 113 

fibroglia, 112 

myoglia, 112 

neuroglia, 112 
Fixing reagents, alcohol, 106 

alcohol-ether for smears, 50 

corrosive alcohol, 115 

formaldehyde, 104 

heat for bacteria, 79 

Marchi's fluid, 117 

Muller's fluid, 117 

Zenker's fluid, 99 
Flagella, stain for, 82 
Flexner's stain for leprosy bacillus, 

116 
Forceps, autopsy, 119 

cover-glass, 43 
Formaldehyde fixation, 104 
Free hydrochloric acid in stomach 

contents, 19 
Freezing microtome, knife for, 91 
Fresh tissue, staining of, 89 
Frozen sections, methods of making, 



Gastric contents, hydrochloric acid 
in, 19 

lactic acid, 21 

microscopic examination of, 18 

pepsin in, 22 

vomitus, 18 
Gastro-intestinal tract, examination 

of, 123 
Gelatin, 76 
Glycerine agar, 75 
Glycerine-albumin fixative, 107 
Glycogen, Best's stain for, 114 
Golgi's stain, 117 
Gonococcus, complement fixation test, 

61 
Gram's iodine, 79 
Gram's staining solution, 79 
Gram- Weigert stain, 114 
Granules in leucocytes, 43, 46, 47 
Guinea-pig, board, 81 

inoculation for tubercle bacilli, 83 

Hair, care of at autopsy, 126 
Hand lotion, 132 
Hanging drop, 50 
Harke's method, 127 
Head-holder, 119 
Heart, opening of, 122 
Heller's test for albumin, 2 
Hematoidin, 30 
Hematoxylin, Delafield's 97, no 

phosphotungstic acid, no, 112 
Hemocytometer, 37 
Hemoglobin, 32 
Hemolysin, anti-sheep, 58 
Hemosiderin, stain for, 113 
Hookworms, 26 
Hyalin in liver cells, in 
Hyalin casts, 17 



Fuchsin, acid-fuchsin, 112 
carbol-fuchsin, 80 

Ganglion cells, stain for, 117 
Gastric contents, amount of, i£ 

blood in, 18 

gross examination of, 18 



Imbedding, celloidin, 91 

paraffin, 102, 105 
Incision, main autopsy, 120 
Inoculation, intravenous rabbit, 84 

subcutaneous guinea-pig, 83 
Instruments, autopsy, 119 
Intestine, opening of, 124 



136 



INDEX 



Iodine, for removing mercury, no 

for Gram's stain, 79 
Iron, test for, 113 

Kidney, examination of, 124 
Knives, autopsy, 119 
microtome, 108 

Labels, protection of, 79 

Lactose bile, yj 

Lactose media, yj 

Leprosy bacilli, 116 

Leucocytes, 42 

Levaditi's stain for treponema, 116 

Lithium carmine, 114 

Litmus milk, 76 

Liver, examination of, 124 

Loeffler's methylene blue, 79 

Lotion, hand, 132 

Lowit's stain for flagella, 82 

Lung, examination of, 122 

Lymphocytes, 43 

Mcjunkin's polychrome stain for 
blood, 50 
for protozoa, 48 
Mcjunkin's tube for blood cultures, 

53 
Malarial organisms, 48 
Mallory's stain for collagen fibrils, 112 
for fibroglia, myoglia and neu- 
roglia fibrils, 112 
eosin-methylene blue for routine 
work, 109 
Mannite media, 77 
Marchi's fluid, 117 
Marrow, examination of, 127 
Mechanical stage, 38 
Megakaryocytes, 48 
Methylene blue, alkaline, in 
Loeffler's, 79 
polychrome, 48 
Methylene blue-eosin stain, Mallory, 

109 
Methyl violet, 79 

Microscopic examination of bacterio- 
logic smears, 79 



Microscopic examination of blood 
films, 41 

feces, 22 

gastric contents, 18 

histologic sections, 88 

sputum, 30 

urine sediment, 13 
Microtome, . celloidin, 93 

freezing, 89 

paraffin, 108 
Microtome knife for celloidin sections, 

93 

for frozen sections, 89 
Middle ear, 127 
Milk, litmus, 76 

bacterial count of milk, 85 
Muscle in feces, 23 
Museum preparations, numbering, 
130 

preserving, 131 
Myelin sheath, staining of, 117 
Myelocytes, 46 
Myoglia fibrils, 112 

Naso-pharnyx, 127 
Neck, block for, 120 
Neuroglia fibrils, 112 
Neutrophils, 43 
Nitrate of silver stain, 116 
Nitric acid for decalcification, 104 
Noguchi's test for globulin, 83 
Nose and throat cultures, 85 

Oil, aniline, 115 

origanum cret. (Merck), 97 
Oxydase, reaction, 43 

Pancreas, examination of, 124 
Paraffin, method of imbedding, 102 
Paraffin microtome, 108 

oven, 106 

sections, staining of, 109 
Parasites, in blood, 48 

in feces, 24 
Pathologic material for bacteriological 
examination, 68 

for immediate diagnosis, 89 



INDEX 



137 



Pathologic material from autopsies, 

118 
from surgical operations, 88 
Pathology, clinical, 88 
Pelvic organs, examination of, 125 
Peptone solution, Dunham's, 77 
Pericardium, opening of, 122 
Petri dishes for plating, 72 
Phenolphthalein as an indicator, 73 
Phosphotungstic acid hematoxylin, 

112 
Phthalein test of kidney function, 9 
Plasma cells in blood, 43 
Plasmodia, 48 
Plate cultures, 72 
Pleural cavities, 122 
Pneumococcus, 82 
Polychrome staining, 48 
Post-mortem examinations, 118 
Protozoa in sections, stain for, 115 
Pure cultures, 72 
Pyronin, 80 

Rabbits for gas bacillus, 84 

for amboceptor, 58 
Reaction of complement fixation in 
gonorrhoea, 61 

of culture media, 73 

Schultze's oxydase, 43 

Wassermann, 55 
Rectum, examination of, 125 
Red blood corpuscles, counting of, 37 

staining of, 41 
Renal casts in urine, 17 
Romanowsky staining, 48 
Round-worms, 26 

Running water for washing specimens, 
102 

Saw, bone, 119 
Scales, for autopsies, 118 
Scalpels, 119 
Scharlach R., 113 
Schering celloidin, 97 
Schultze's oxydase reaction, 43 
Sedimenting of tubercle bacilli, 78 
Serum for amboceptor, 58 



Serum for opsonic test, 50 
for Wassermann, 55 
for Widal reaction, 50 
Silver stain for spirochetes, 116 
Slides, cleaning of, 41 
Smears of blood, staining of, 41 
Specific gravity of urine, 1 
Spirals, Curschmann's in sputum, 30 
Spirochasta pallida, Levaditi stain for, 
116 

Mcjunkin's stain for, 48 
Spleen, examination of, 123 
Spores, staining of, 82 
Sputum, blood in, 30 

caseous masses in, 29 

cells in, 30 

Charcot-Leyden crystals in, 30 

cultivation of tubercle bacilli 
from, 78 

Dittrich's plugs in, 30 

elastic fibrils in 30 

macroscopic appearance of, 29 

microscopic examination of, 30 
Staining tubercle bacilli in, 30 
Staining dishes for polychrome stain- 
ing, 115 

for celloidin sections, 101 

for paraffin sections, 1 1 1 
Stains, alum carmine, 114 

aniline blue, Mallory, 112 

Best's carmine for, 114 

carbol-fuchsin, 80 

Czaplewsky fluid, 81 

Delafield's hematoxylin, 97 

differentiation of, 109 

eosin-methylene blue, 109 

for bacteria in tissue, 115 

for blood, 41 

for calcium, 114 

for collagen fibrils, 112 

for elastic fibrils, 113 

for fat, 113 

for fibrin, 114 

for fibroglia fibrils, 112 

for flagella, 82 

for frozen sections, 89, 97 

for glycogen, 114 



138 



INDEX 



Stains for iron, 113 

for leprosy bacilli, 116 

for malarial parasites, 48 

for myelin sheath, 117 

for myoglia fibrils, 112 

for neuroglia fibrils, 112 

for protozoa in sections, 115 

for spirochetes, 48 

for spores, 82 

for tubercle bacillus in sections, 

116 
for tubercle bacillus in smears, 80 
Golgi's stain, 117 
Gram's iodine, 80 

staining solution, 79 
Gram-Weigert stain, 114 
Hematoxylin, alum, 97 

Mallory's phosphotungstic 
acid, 112 
iodine, Gram's, 79 
Levaditi, 114 

Loeffier's methylene blue, 79 
Lowit's for flagella, 82 
Mcjunkin's polychrome stain for 
blood, 50 

for protozoa, 48 
Mallory's eosin-methylene blue, 
109 

aniline blue, 112 

phosphotungstic acid hema- 
toxylin, 112 
Marchi's stain, 117 
methylene blue alkaline, 1 1 1 

polychrome, 48 

Loeffler's, 79 
methyl violet for, 79 
phosphotungstic acid hematoxy- 
lin, Mallory, 112 
polychrome, 48 
Romanowsky, 48 
Scharlach R., for fat, 113 
Silver for spirochetes, 116 
Verhoefl's for elastic fibrils, 113 
Weigert-Gram, 114 
Ziehl-Neelsen, 80 
Sterilization, dry heat, 68 
steam, 75 . 



Sterilization, steam under pressure, 75 
Stomach contents {see gastric con- 
tents) . 
Stomach, examination of, 124 
Swabs, sterile for cultures, 68 
Syphilis, Wassermann reaction for, 55 

Tenia nana, 28 

saginata, 28 

solium, 28 
Testicles, examination of, 125 
Test-tubes, 2 
Tetanus, diagnosis of, 84 
Tissue, autopsy, 118 

surgical, 88 
Titration of culture media, 73 

of gastric contents, 20 

in nitrogen determinations, 1 1 
Transplanting cultures, J2 
Treponema pallidum, 116 
Trichinella spiralis, 28 
Tubercle bacillus, cultivation of, from 
sputum, 78 

in feces, 24 

in sputum, 30 

in tissue, 116 

in urine, 83 
Typhoid bacillus, 24, yy 

Urine, acetone in, 7 

ammonia nitrogen, 13 

albumin in, 1 

albumose in, 3 

Bence-Jones body in, 3 

bile pigment in, 8 

blood in, 17 

casts in, 17 

chlorides in, 9 

collection of, 1 

diacetic acid in, 8 

diazo reaction in, 8 

Esbach test, 3 

glucose in, 4 

heat and acetic acid test, 2 

Heller's test, 2 

indican in, 9 

mucin in, 17 



INDEX 



139 



Urine, occult blood, 24 

organized sediment, 16 
oxybutyric acid in, 8 
phosphates in, 16 
phthalein test for renal function, 

9 

resins in, 3 

unorganized sediment of acid 

urine, 14 
unorganized sediment of alkaline 

urine, 16 

urates in, 14 

ureometer, 1 

uric acid, 13 

Uterine curettings. 93 



Wassermann reaction, amboceptor 
for, 58 

antigen for, 59 

complement for, 58 

corpuscles for, 63 

method of making the test, 65 

patients serum for, 56 

reporting results of tests, 55 

saline for, 64 
Weigert-Gram stain, 114 
Widal reaction, 50 
Wood stain, 89 
Worms in feces, 26 

Xylol-colophonium, 97 



Vaccines, bacterial, 87 
Verhoeff's stain for elastic fibrils, 



113 



Zenker's fluid, 99 
Ziehl-Xeelsen carbol-fuchsin, 80 



